JP2019522466A - Mutant PD-L1 polypeptide, T cell regulatory multimeric polypeptide, and methods of use thereof - Google Patents

Abstract

The present disclosure provides a fusion polypeptide comprising a mutant PD-L1 immunoregulatory polypeptide and a mutant immunomodulatory peptide. The present disclosure provides T cell regulatory multimeric polypeptides and compositions comprising the same, and T cell regulatory multimeric polypeptides include the mutant immunomodulatory polypeptides of the present disclosure. The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a T cell regulatory multimeric polypeptide and a host cell comprising the nucleic acid. The present disclosure provides a method of modulating the activity of a T cell, the method comprising contacting the T cell with a T cell modulating multimeric polypeptide of the present disclosure.

Description

  The adaptive immune response is expressed by antigen-presenting cells by the T cell receptor (TCR) present on the surface of the T cells and the major histocompatibility antigen complex (MHC: also called human leukocyte antigen (HLA) complex in humans). It involves binding to small peptide antigens that exist non-covalently on the surface of (APC). This binding corresponds to the targeting mechanism of the immune system and is a molecular interaction required for T cell regulation (activation or suppression) and effector function.

  Following epitope-specific cell targeting, targeted T cells are activated via the binding of costimulatory proteins found in APC and the corresponding costimulatory proteins of T cells. To induce T cell specificity and activation or suppression, signals of both epitope / TCR binding and binding of APC costimulatory proteins to T cell costimulatory proteins are required. Although TCR is specific for a given epitope, costimulatory proteins are not epitope specific and instead are generally expressed on all T cells or large T cell subsets.

  The present disclosure provides a mutant PD-L1 immunomodulatory polypeptide and a fusion polypeptide comprising the mutant immunomodulatory peptide. The present disclosure provides a T cell regulatory multimeric polypeptide and a composition comprising a T cell regulatory multimeric polypeptide, wherein the T cell regulatory multimeric polypeptide comprises a mutant immunomodulatory polypeptide of the disclosure. The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a T cell regulatory multimeric polypeptide and a host cell comprising the nucleic acid. The present disclosure provides a method of modulating the activity of a T cell, the method comprising contacting the T cell with a T cell modulating multimeric polypeptide of the present disclosure.

Figure 3 schematically illustrates various embodiments of the T cell regulatory multimeric polypeptides of the present disclosure. In such embodiments, disulfide bonds are formed between MHC (eg, HLA) polypeptides present in separate polypeptides. The amino acid sequence (A) of wild type mouse PD-L1 polypeptide and the amino acid sequence (B) of wild type human PD-L1 polypeptide are shown. 2 shows a sequence alignment (C) of mouse and human PD-L1 amino acid sequences. Examples of mutant PD-L1 polypeptides (DG) are shown. Examples of mutant PD-L1 polypeptides (HK) are shown. Examples of mutant PD-L1 polypeptides (LM) are shown. The amino acid sequence (A) of mouse PD-1 and the amino acid sequence (B) of human PD-1 are shown. The amino acid sequence (C) of mouse B7-1 and the amino acid sequence (D) of human B7-1 are shown. 1 shows the amino acid sequence (A) of an immunoglobulin Fc polypeptide. 1 shows the amino acid sequence (B) of an immunoglobulin Fc polypeptide. 1 shows the amino acid sequence (C) of an immunoglobulin Fc polypeptide. 1 shows the amino acid sequence (A and B) of human leukocyte antigen (HLA) class I heavy chain polypeptide. The signal sequence is underlined. 1 shows the amino acid sequence (C) of human leukocyte antigen (HLA) class I heavy chain polypeptide. The signal sequence is underlined. Homo sapiens (NP_004039.1: SEQ ID NO: 3), Pan trologites (NP_0010090666.1: SEQ ID NO: 4), Macaca mulatta (NP_00104062.1: SEQ ID NO: 5), Bos taurus (NP_76638.1: SEQ ID NO: 6), and us FIG. 5 shows an alignment of multiple amino acid sequences of a beta-2 microglobulin (β2M) precursor (ie, including a leader sequence) derived from musculus (NP — 03865.2: SEQ ID NO: 7). Amino acids 1-20 are signal peptides. Screening of PD-L1 mutants using a high-throughput microbead binding assay by FACS (A and B) and microbead binding data by FACS for PD-L1 mutants (C) are shown. Characterization of PD-L1 variants with altered binding to PD-1 or B7-1 is shown. Shows that PD-1 competes with B7-1 for binding to PD-L1. Table 1 is shown. Table 2 is shown. Figure 2 shows the in vivo effect of PD-L1 / synTac on pathogenic epitope-specific CD8 ⁺ T cells.

Definitions The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and refer to a polymeric form of nucleotides, either ribonucleotides or deoxyribonucleotides, of any length. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, a hybrid of DNA and RNA, or a polymer containing purine and pyrimidine bases, or Includes polymers comprising other natural nucleotide bases, chemically or biochemically modified nucleotide bases, non-natural nucleotide bases, or derivatized nucleotide bases.

  The terms “peptide”, “polypeptide”, and “protein” are used interchangeably herein to refer to encoded and non-encoded amino acids that are chemically or biochemically modified or derivatized amino acids. The term refers to a polymerized amino acid of any length, which may be included, and a polypeptide having a modified peptide backbone.

  A polynucleotide or polypeptide has a certain percentage of “sequence identity” with respect to another polynucleotide or polypeptide, which is the percentage when two sequences are aligned and compared. Means that the bases or amino acids are the same and exist at the same relative position. Sequence identity can be determined in many different ways. To determine sequence identity, various convenient methods and computer programs (eg, BLAST, T-COFFEE, MUSCLE, MATFT, etc.) can be used to align sequences, such methods and computer programs are , Ncbi. nlm. nili. gov / BLAST, ebi. ac. uk / Tools / msa / toffee /, ebi. ac. uk / Tools / msa / mouse /, mart. cbrc. Available through the World Wide Web at several sites, including jp / alignment / software /. See, for example, Altschul et al. (1990), J.A. Mol. Bioi. 215: 403-10.

  The term “conservative amino acid substitution” refers to the interchangeability in a protein of amino acid residues having similar side chains. For example, a group of amino acids having an aliphatic side chain consists of glycine, alanine, valine, leucine, and isoleucine, and a group of amino acids having an aliphatic-hydroxyl side chain consists of serine and threonine, and has an amide-containing side chain. A group of amino acids having asparagine and glutamine, a group of amino acids having aromatic side chains consisting of phenylalanine, tyrosine, and tryptophan, and a group of amino acids having basic side chains from lysine, arginine, and histidine. Thus, a group of amino acids having acidic side chains consists of glutamic acid and aspartic acid, and a group of amino acids having sulfur-containing side chains consists of cysteine and methionine. Examples of conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine-glutamine.

“Binding” as used herein (eg, with respect to binding of a T cell regulatory multimeric polypeptide of the present disclosure to a polypeptide (eg, a T cell receptor) present on a T cell) is mediated non-covalent binding. Refers to the interaction. A binding interaction is generally characterized by a dissociation constant (K _D ) of less than 10 ⁻³ M. Preferred K _D values are less than 10 ⁻⁶ M, less than 10 ⁻⁷ M, less than 10 ⁻⁸ M, less than 10 ⁻⁹ M, less than 10 ⁻¹⁰ M, less than 10 ⁻¹¹ M, less than 10 ⁻¹² M, 10 ⁻ Less than ¹³ M, less than 10 ⁻¹⁴ M, or less than 10 ⁻¹⁵ M. "Affinity" refers to the bond strength, increased binding affinity is correlated with a decrease in K _D.

  As used herein, the term “immunological synapse” or “immune synapse” generally refers to the natural interface between two immune cells that interact in an adaptive immune response, such as, for example, , An interface between antigen presenting cells (APC) or target cells and effector cells (eg, lymphocytes, effector T cells, natural killer cells, and the like). Immunological synapses between APCs and T cells are generally initiated by the interaction of T cell antigen receptors with major histocompatibility complex molecules. For this, see, for example, Bromley et al. , Annu Rev Immunol. 2001; 19: 375-96, the disclosure content of which is incorporated herein by reference in its entirety.

“T cells” are all types of CD3 expressing, including helper T cells (CD4 ⁺ cells), cytotoxic T cells (CD8 ⁺ cells), regulatory T cells (Treg), and NK-T cells. Contains immune cells.

  As used herein, the term “costimulatory polypeptide” refers to a primary signal (eg, a TCR / CD3 complex and a peptide by specifically binding to a co-stimulatory polypeptide present on a T cell. In addition to major histocompatibility complex (MHC) polypeptides loaded with T cell responses (including but not limited to proliferation, activation, differentiation, and the like). Polypeptides present in antigen presenting cells (APCs) (eg, dendritic cells, B cells, and the like) that generate mediating signals.

  The “regulatory domain” of a T cell regulatory multimeric polypeptide of the present disclosure includes a costimulatory polypeptide.

  As used herein, “heterologous” refers to nucleotides or polypeptides that are not found in natural nucleic acids or proteins, respectively.

  As used herein, “recombinant” results in a construct in which a particular nucleic acid (DNA or RNA) has a structural coding or non-coding sequence that is distinguishable from endogenous nucleic acids found in the natural system. It is meant to be the product of various combinations of cloning, restriction, polymerase chain reaction (PCR) and / or ligation steps. A DNA sequence encoding a polypeptide can be constructed from a cDNA fragment or a series of syntheses to produce a synthetic nucleic acid capable of expression from a recombinant transcription unit included in a cell or cell-free transcription and translation system. Can be constructed from oligonucleotides.

  The terms “recombinant expression vector” or “DNA construct” are used interchangeably herein to refer to a DNA molecule comprising a vector and one insert. Recombinant expression vectors are usually generated for the purpose of expression and / or amplification of the insert (s) or construction of other recombinant nucleotide sequences. The insert fragment (s) may or may not be operably linked to a promoter sequence and may or may not be operably linked to a DNA regulatory sequence.

  For example, when exogenous DNA, such as a recombinant expression vector, has been introduced inside a cell, the cell has been “genetically modified” or “transformed” or “gene introduced” by such DNA. In the presence of exogenous DNA, persistent or transient genetic changes occur. The transforming DNA may or may not be integrated into the cell's genome (covalent ligation). In prokaryotes, yeast, and mammalian cells, for example, transforming DNA can be maintained with episomal elements such as plasmids. With respect to eukaryotic cells, a stably transformed cell is a cell in which the transforming DNA is integrated into the chromosome so that the transforming DNA is inherited by daughter cells through chromosome replication.

  As used herein, “host cell” refers to an eukaryotic cell in vivo or in vitro, or a cell derived from a multicellular organism (eg, a cell line) cultured as a unicellular entity, wherein the eukaryotic cell is a nucleic acid. (Eg, an expression vector comprising a nucleotide sequence encoding a multimeric polypeptide of the present disclosure) that can be used or has been used, and has been genetically modified by the nucleic acid Cell progeny. It is understood that unicellular progeny are naturally, accidental, or deliberately mutated, so that the morphology or genomic DNA complement or total DNA complement cannot necessarily be completely identical to the original parent. A “recombinant host cell” (also referred to as “genetically modified host cell”) is a host cell into which a heterologous nucleic acid such as an expression vector has been introduced. For example, a genetically modified eukaryotic host cell is genetically modified, for example, a foreign nucleic acid that is foreign to the eukaryotic host cell, or a recombinant nucleic acid that is not normally found in the eukaryotic host cell, etc. The heterologous nucleic acid has been introduced into a suitable eukaryotic host cell.

The terms “treatment”, “treating” and the like are generally used herein to mean expression of a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or its symptoms and / or therapeutic in terms of partially or completely curing the disease and / or adverse effects that may result from the disease. It can be. “Treatment” as used herein includes any treatment of a disease or condition in an animal and (a) may be susceptible to, but still diagnosed as having, the disease or condition. Prevention of the onset of a disease or symptom in a non-subject, (b) suppression of the disease or symptom, ie, suppression of its onset, or (c) reduction of the disease, ie, induction of regression of the disease. The therapeutic agent may be administered before, during, or after the occurrence of the disease or injury. The treatment of ongoing diseases is of particular interest, in which the treatment stabilizes or reduces undesirable clinical symptoms in the patient. Such treatment is preferably performed before the function in the affected tissue is completely lost. The subject's treatment will desirably be administered during the symptom stage of the disease, and possibly after the symptom stage of the disease.

  The terms “individual”, “subject”, “host”, and “patient” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired. Mammals include, for example, humans, non-human primates, rodents (eg, rats, mice), rabbits (eg, rabbits), ungulates (eg, cows, sheep, pigs, horses, goats, and The same).

  Before the additional description of the invention, it is to be understood that the invention is not limited to the specific embodiments described, and thus embodiments may of course vary. The terminology used herein is for the purpose of describing particular embodiments only, and the scope of the present invention will be limited only by the claims that follow. It will also be understood that no limitation by the terminology used is intended.

  Where a range of values is given, unless stated otherwise in context, each intervening value up to one-tenth of the lower limit, between the upper limit and lower limit of the range, and the stated range Any other stated or intervening value is understood to be encompassed by the present invention. Such upper and lower limits of a smaller range can be independently included in the smaller range, and these upper and lower limits are also included in the present invention and specifically excluded in the described range. It is subject to any limit value. Where the stated range includes one or both of the limit values, ranges excluding either or both of those included limit values are also included in the invention.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned in this specification are herein incorporated by reference to disclose and explain the methods and / or materials with which the publications are cited.

  It is noted that the singular forms “a”, “an”, and “the”, as used in the specification and the appended claims, include plural referents unless the context clearly indicates otherwise. must not. Thus, for example, reference to “PD-L1 variant” includes a plurality of such variants, and a reference to “HLA polypeptide” refers to one or more HLA polypeptides, and those known to those of skill in the art Including references to equivalent forms, etc. It is further noted that the claims may be drafted excluding any optional elements. Accordingly, this description serves as a preliminary basis for the use of exclusive terms such as “simply”, “only”, and the like in connection with the claim element description, or for the use of “negative” limitations. Is intended.

  Certain features of the invention are described in connection with separate embodiments for clarity, but it will be understood that they may be provided in combination in a single embodiment. Conversely, various features of the invention are described in connection with a single embodiment for simplicity, but may be provided separately or in any appropriate subcombination. . All combinations of embodiments relating to the present invention are expressly encompassed by the present invention, and each and every combination is disclosed herein as if individually and explicitly disclosed. Moreover, all of the various embodiments and subcombinations of elements thereof are also expressly encompassed by the present invention, as each and every such subcombination is individually and explicitly disclosed herein. Is disclosed herein.

  The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, and the actual publication dates may need to be individually confirmed.

DETAILED DESCRIPTION The present disclosure provides a mutant immunomodulatory polypeptide and a fusion polypeptide comprising the mutant immunomodulatory peptide. The present disclosure provides a T cell regulatory multimeric polypeptide and a composition comprising a T cell regulatory multimeric polypeptide, wherein the T cell regulatory multimeric polypeptide comprises a mutant immune regulatory polypeptide of the present disclosure. The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a T cell regulatory multimeric polypeptide and a host cell comprising the nucleic acid. The present disclosure provides a method of modulating the activity of a T cell, the method comprising contacting the T cell with a T cell modulating multimeric polypeptide of the present disclosure.

  The T cell regulatory multimeric polypeptides of the present disclosure are also referred to as “synTac polypeptides”. The synTac polypeptides of the present disclosure include a mutated regulatory domain that is compared to the affinity of the wild-type PD-L1 regulatory domain for an immunomodulatory polypeptide (eg, an immunomodulatory polypeptide present in T cells). A decrease in binding affinity for an immunomodulatory polypeptide (eg, PD-1 or B7-1). The disclosed synTac polypeptides can modulate (eg, suppress) the activity of target T cells. The disclosed synTac polypeptides increase the specificity of the target cell.

Mutant immunomodulatory polypeptides The present disclosure provides mutant PD-L1 regulatory polypeptides. The wild type amino acid sequence of human PD-L1 is shown in FIG. 2A.

  Wild type PD-L1 binds to PD1 and B7-1. The amino acid sequence of mouse PD-1 is shown in FIG. 3A, and the amino acid sequence of human PD-1 is shown in FIG. 3B. The amino acid sequence of mouse B7-1 is shown in FIG. 3C, and the amino acid sequence of human B7-1 is shown in FIG. 3D. In some cases, a mutant PD-L1 polypeptide of the present disclosure binds to PD-1 with reduced affinity compared to wild-type PD-L1 binding to PD1. In some cases, a mutant PD-L1 polypeptide of the present disclosure binds to B7-1 with lower affinity compared to binding of wild-type PD-L1 to B7-1. In some cases, a mutant PD-L1 polypeptide of the present disclosure binds to PD-1 with substantially the same affinity as the binding affinity of wild-type PD-L1 to PD-1, and wild-type to B7-1. It binds to B7-1 with lower affinity compared to the binding of PD-L1. In some cases, a mutant PD-L1 polypeptide of the present disclosure binds to PD-1 with low affinity compared to wild-type PD-L1 binding to PD1 and wild-type PD-L1 binding to B7-1. Binds to B7-1 with lower affinity than In some cases, a mutant PD-L1 polypeptide of the present disclosure binds to PD-1 with low affinity compared to wild-type PD-L1 binding to PD-1, and wild-type PD-L1 to B7-1. Binds to B7-1 with substantially the same affinity as

  In some cases, a mutant PD-L1 polypeptide of the present disclosure is a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2A relative to PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3A). It shows a decrease in binding affinity for PD-1 compared to the binding affinity of the peptide. For example, in some cases, a mutant PD-L1 polypeptide of the present disclosure can have a binding affinity of a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2A to a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3A. It binds to PD-1 with a lower binding affinity compared to. For example, in some cases, a mutant PD-L1 polypeptide of the present disclosure comprises a PD- comprising the amino acid sequence shown in FIG. 2A relative to PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3A). At least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% lower than at least the binding affinity of the L1 polypeptide, 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or greater than 95% lower It binds to PD-1 with affinity.

  In some cases, a mutant PD-L1 polypeptide of the present disclosure is a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2B relative to PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3B). It shows a decrease in binding affinity for PD-1 compared to the binding affinity of the peptide. For example, in some cases, a mutant PD-L1 polypeptide of the present disclosure can have a binding affinity of a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2B to a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3B. It binds to PD-1 with a lower binding affinity compared to. For example, in some cases, a mutant PD-L1 polypeptide of the present disclosure comprises a PD- comprising the amino acid sequence shown in FIG. 2B relative to PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3B). At least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% lower than at least the binding affinity of the L1 polypeptide, 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or greater than 95% lower It binds to PD-1 with affinity.

In some cases, the wild type mouse PD-L1 ectodomain comprises the following amino acid sequence:
FT ITAPKDLYVV EYGSNVTMEC RFPVERELDL LALVVYWEKE DEQVIQFVAG EEDLKPQHSN FRGRASLPKD QLLKGNAALQ ITDVKLQDAG VYCCIISYGG ADYKRITLKV NAPYRKINQR ISVDPATSEH ELICQAEGYP EAEVIWTNSD HQPVSGKRSV TTSRTEGMLL NVTSSLRVNA TANDVFYCTF WRSQPGQNHT AELIIPELPA THPPQNR (SEQ ID NO: 1).

In some cases, the wild type human PD-L1 ectodomain comprises the following amino acid sequence:
FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKI (SEQ ID NO: 2).

  In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises a PD-L1 comprising the amino acid sequence set forth in SEQ ID NO: 1 for PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3A). It shows a decrease in binding affinity for PD-1 compared to the binding affinity of the polypeptide. For example, in some cases, a mutant PD-L1 polypeptide of the present disclosure can have a binding affinity of a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 for a PD-1 polypeptide comprising the amino acid sequence set forth in FIG. 3A. It binds to PD-1 with a lower binding affinity compared to sex. For example, in some cases, a mutant PD-L1 polypeptide of the present disclosure comprises a PD comprising the amino acid sequence set forth in SEQ ID NO: 1 for PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3A). -At least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% lower compared to the binding affinity of the L1 polypeptide; At least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or more than 95% lower It binds to PD-1 with binding affinity.

  In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises a PD-L1 comprising the amino acid sequence set forth in SEQ ID NO: 2 relative to PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3B). It shows a decrease in binding affinity for PD-1 compared to the binding affinity of the polypeptide. For example, in some cases, a mutant PD-L1 polypeptide of the present disclosure can have a binding affinity of a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 for a PD-1 polypeptide comprising the amino acid sequence set forth in FIG. 3B. It binds to PD-1 with a lower binding affinity compared to sex. For example, in some cases, a mutant PD-L1 polypeptide of the present disclosure comprises a PD comprising the amino acid sequence set forth in SEQ ID NO: 2 relative to PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3B). -At least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% lower compared to the binding affinity of the L1 polypeptide; At least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or more than 95% lower It binds to PD-1 with binding affinity.

  In some cases, a mutant PD-L1 polypeptide of the present disclosure exhibits reduced binding affinity for PD-1, as described above, and the binding affinity of the wild type PD-L1 polypeptide for the wild type B7-1 polypeptide. Retain at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the sex. For example, in some cases, a mutant PD-L1 polypeptide of the present disclosure comprises a PD comprising the amino acid sequence set forth in SEQ ID NO: 2 relative to PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3B). A reduced binding affinity for PD-1 compared to the binding affinity of the L1 polypeptide, and a wild type B7-1 polypeptide (eg, a B7-1 polypeptide comprising the amino acid sequence shown in FIG. 3D) At least 80%, at least 85%, at least 90%, at least 95%, at least 98 of the binding affinity of a wild type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2) %, Or at least 99%.

  In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises a PD-L1 comprising the amino acid sequence set forth in SEQ ID NO: 2 relative to PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3B). The binding affinity for the PD-1 polypeptide is reduced by about 40% to about 60% compared to the binding affinity of the polypeptide, and the wild type B7-1 polypeptide (eg, the amino acid sequence shown in FIG. 3D) At least 80%, at least 85%, at least 90% of the binding affinity of a wild type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2). %, At least 95%, at least 98%, or at least 99%.

  In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises a PD-L1 comprising the amino acid sequence set forth in SEQ ID NO: 1 for PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3A). Compared to the binding affinity of the polypeptide, the binding affinity for the PD-1 polypeptide is reduced by about 40% to about 60%, and the wild type B7-1 polypeptide (eg, the amino acid sequence shown in FIG. 3C) At least 80%, at least 85%, at least 90% of the binding affinity of a wild type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1). %, At least 95%, at least 98%, or at least 99%.

  In some cases, a mutant PD-L1 polypeptide of the present disclosure has a binding affinity for PD-1 of 1 nM to 1 mM. In some cases, a mutant PD-L1 polypeptide of the present disclosure has a binding affinity for PD-1 of 100 nM to 100 μM. As another example, in some instances, a mutant PD-L1 polypeptide of the present disclosure has about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to PD1 of about 10 μM, about 10 μM to about 15 μM, about 15 μM to about 20 μM, about 20 μM to about 25 μM, about 25 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM (for example, the amino acids shown in FIG. 3 Binding affinity for PD1 polypeptide containing sequence) Have

  A mutant PD-L1 polypeptide of the present disclosure is compared to a wild-type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2). May have a single amino acid substitution. In some cases, a mutant PD-L1 polypeptide of the present disclosure is a wild-type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence set forth in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2). ) With 2 to 10 amino acid substitutions. In some cases, a mutant PD-L1 polypeptide of the present disclosure is a wild-type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence set forth in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2). ) And two amino acid substitutions. In some cases, a mutant PD-L1 polypeptide of the present disclosure is a wild-type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence set forth in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2). ) And 3 amino acid substitutions. In some cases, a mutant PD-L1 polypeptide of the present disclosure is a wild-type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence set forth in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2). ) And 4 amino acid substitutions. In some cases, a mutant PD-L1 polypeptide of the present disclosure is a wild-type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence set forth in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2). ) And 5 amino acid substitutions. In some cases, a mutant PD-L1 polypeptide of the present disclosure is a wild-type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence set forth in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2). ) And 6 amino acid substitutions. In some cases, a mutant PD-L1 polypeptide of the present disclosure is a wild-type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence set forth in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2). ) With 7 amino acid substitutions. In some cases, a mutant PD-L1 polypeptide of the present disclosure is a wild-type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence set forth in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2). ) And 8 amino acid substitutions. In some cases, a mutant PD-L1 polypeptide of the present disclosure is a wild-type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence set forth in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2). ) And 9 amino acid substitutions. In some cases, a mutant PD-L1 polypeptide of the present disclosure is a wild-type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence set forth in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2). ) And 10 amino acid substitutions.

  The mutant PD-L1 polypeptides of the present disclosure can have a length of 200-240 amino acids. For example, in some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid length of 200-220, or 220-240 amino acids. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid length of 200-219. In some cases, a mutant PD-L1 polypeptide of the present disclosure has a length of 219 amino acids.

D26 Substitution In some cases, a variant PD-L1 polypeptide of the present disclosure exhibits at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. The amino acid 26 is an amino acid other than aspartic acid. For example, the amino acid 26 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 26 is Ala, Gly, Val, Leu, or Ile. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 26 is Ala, Gly, Val, Leu, Ile, or Arg. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 26 is Ala. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 26 is Gly. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 26 is Val. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 26 is Leu. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 26 is Ile. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 26 is Arg. In some cases, the mutant PD-L1 polypeptide comprises a PD comprising the amino acid sequence shown in FIG. 2B (or SEQ ID NO: 2) relative to PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3B). -Shows about 40% to about 60% reduction in binding affinity for PD-1 polypeptide compared to the binding affinity of L1 polypeptide, as shown in wild-type B7-1 polypeptide (eg, shown in FIG. 3D At least 80% of the binding affinity of a wild-type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2B or SEQ ID NO: 2) to a B7-1 polypeptide comprising an amino acid sequence), at least Retain 85%, at least 90%, at least 95%, at least 98%, or at least 99%.

  In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B with an amino acid substitution of D26. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2 with a D8 amino acid substitution. For example, in some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 26 is any amino acid other than aspartic acid, eg, amino acid 26 is Gly, Ala , Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 26 is Ala, Gly, Val, Leu, or Ile. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, and amino acid 26 is Ala, Gly, Val, Leu, Ile, or Arg. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 26 is Ala instead of Asp. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 26 is Val instead of Asp. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 26 is Leu instead of Asp. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, and amino acid 26 is Gly instead of Asp. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 26 is Ile instead of Asp. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 26 is Arg instead of Asp.

  In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2 with a D8 amino acid substitution. For example, in some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, wherein amino acid 8 is any amino acid other than aspartic acid, for example, amino acid 8 is Gly, Can be Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 8 is Ala, Gly, Val, Leu, or Ile instead of Asp. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 8 is Ala, Gly, Val, Leu, Ile, or Arg instead of Asp. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 8 is Ala instead of Asp. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 8 is Val instead of Asp. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 8 is Leu instead of Asp. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 8 is Gly instead of Asp. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 8 is Ile. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 8 is Arg instead of Asp.

  In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2D. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2E. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2F. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2G.

Substitution of T37 In some cases, a mutant PD-L1 polypeptide of the present disclosure has at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. The amino acid 37 is an amino acid other than threonine. For example, the amino acid 37 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln. Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 37 is Gly, Ala, Val, Leu, Ile, Arg, Lys, or His. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 37 is Arg, Lys, or His. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 37 is Gly, Ala, Val, Leu, or Ile. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 37 is Arg. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 37 is Lys. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 37 is His. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 37 is Gly. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 37 is Ala. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 37 is Val. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 37 is Leu. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 37 is Ile. In some cases, the mutant PD-L1 polypeptide has an amino acid sequence shown in FIG. 2B (or SEQ ID NO: 2) relative to PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3B). A binding affinity of about 15% to about 35% of the binding affinity exhibited by the containing PD-L1 polypeptide is shown for the PD-1 polypeptide and the wild type B7-1 polypeptide (eg, the amino acid shown in FIG. 3D). Compared to the binding affinity of a wild-type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 3B or SEQ ID NO: 2) to a B7-1 polypeptide comprising the sequence). A decrease in binding affinity for 1 (eg, a decrease in binding affinity for B7-1 of about 70% to about 90%).

  In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B with an amino acid substitution of T37. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2 with a T19 amino acid substitution. For example, in some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 37 is any amino acid other than threonine, eg, amino acid 37 is Gly, Ala, It can be Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 37 is Gly, Ala, Val, Leu, Ile, Arg, His, or Lys instead of Thr. is there. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 37 is Gly, Ala, Val, Leu, or Ile instead of Thr. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 37 is Arg, His, or Lys instead of Thr. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 37 is Arg instead of Thr. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 37 is Lys instead of Thr. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 37 is His instead of Thr. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 37 is Gly instead of Thr. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 37 is Ala instead of Thr. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 37 is Val instead of Thr. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 37 is Leu instead of Thr. In some instances, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 37 is Ile instead of Thr.

  In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2 with a T19 amino acid substitution. For example, in some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in SEQ ID NO: 2, wherein amino acid 19 is any amino acid other than threonine, for example, amino acid 19 is Gly, Ala , Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, wherein amino acid 19 is Gly, Ala, Val, Leu, Ile, Arg, His, or Lys instead of Thr. It is. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 19 is Gly, Ala, Val, Leu, or Ile instead of Thr. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 19 is Arg, His, or Lys instead of Thr. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, wherein amino acid 19 is Arg instead of Thr. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, wherein amino acid 19 is Lys instead of Thr. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, wherein amino acid 19 is His instead of Thr. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 19 is Gly instead of Thr. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 19 is Ala instead of Thr. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, wherein amino acid 19 is Val instead of Thr. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, wherein amino acid 19 is Leu instead of Thr. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, wherein amino acid 19 is Ile instead of Thr.

In some instances, a mutant PD-L1 polypeptide of the present disclosure has at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. The amino acid 54 is an amino acid other than isoleucine. For example, the amino acid 54 is Gly, Ala, Val, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln. Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. Amino acid 54 is an amino acid other than isoleucine or valine, for example, amino acid 54 is Gly, Ala, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 54 is Ala, Gly, Leu, Glu, or Asp. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 54 is Glu or Asp. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 54 is Ala. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 54 is Gly. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 54 is Leu. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 54 is Asp. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 54 is Glu. In some cases, the mutant PD-L1 polypeptide has the amino acid sequence shown in FIG. 2B (or SEQ ID NO: 2) relative to PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 2B). A binding affinity of about 70% to about 100% of the binding affinity exhibited by the containing PD-L1 polypeptide is shown for the PD-1 polypeptide and the wild type B7-1 polypeptide (eg, the amino acid shown in FIG. 3D). Compared to the binding affinity of a wild-type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 3B or SEQ ID NO: 2) to a B7-1 polypeptide comprising the sequence). A decrease in binding affinity for 1 (eg, a decrease in binding affinity for B7-1 of about 40% to about 90%).

  In some cases, a variant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2A Amino acid 54 is an amino acid other than valine, for example, amino acid 54 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a variant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2A Amino acid 54 is an amino acid other than isoleucine or valine, for example, amino acid 54 is Gly, Ala, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a variant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2A And amino acid 54 is Ala, Gly, Leu, Glu, or Asp. In some cases, a variant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2A And amino acid 54 is Glu or Asp. In some cases, a variant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2A And amino acid 54 is Ala. In some cases, a variant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2A And amino acid 54 is Gly. In some cases, a variant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2A And amino acid 54 is Leu. In some cases, a variant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2A And amino acid 54 is Asp. In some cases, a variant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2A And amino acid 54 is Glu. In some cases, the mutant PD-L1 polypeptide has the amino acid sequence shown in FIG. 2A (or SEQ ID NO: 1) relative to PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3A). A binding affinity of about 70% to about 100% of the binding affinity exhibited by the containing PD-L1 polypeptide is shown for the PD-1 polypeptide and the wild type B7-1 polypeptide (eg, the amino acid shown in FIG. 3C). Compared to the binding affinity of a wild-type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2A or SEQ ID NO: 1) to a B7-1 polypeptide comprising the sequence) A decrease in binding affinity for 1 (eg, a decrease in binding affinity for B7-1 of about 40% to about 90%).

  In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B with an amino acid substitution of I54. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2 having an amino acid substitution of I36. For example, in some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, amino acid 54 is any amino acid other than isoleucine, for example, amino acid 54 is Gly, Ala, Can be Val, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 54 is any amino acid other than isoleucine or valine, eg, amino acid 54 is Gly, Ala, It can be Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, and amino acid 54 is Ala, Gly, Leu, or Asp instead of Ile. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 54 is Ala instead of Ile. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 54 is Leu instead of Ile. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 54 is Gly instead of Ile. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 54 is Asp instead of Ile.

  In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2A with an amino acid substitution of V54. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 1 with an amino acid substitution of V36. For example, in some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2A, wherein amino acid 54 is any amino acid other than valine, eg, amino acid 54 is Gly, Ala, It can be Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2A, wherein amino acid 54 is any amino acid other than isoleucine or valine, eg, amino acid 54 is Gly, Ala, It can be Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2A, and amino acid 54 is Ala, Gly, Leu, Glu, or Asp instead of Val. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2A, wherein amino acid 54 is Glu or Asp instead of Val. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2A, wherein amino acid 54 is Ala instead of Val. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2A, wherein amino acid 54 is Leu instead of Val. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2A, wherein amino acid 54 is Gly instead of Val. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2A, wherein amino acid 54 is Asp instead of Val. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2A, wherein amino acid 54 is Glu instead of Val.

  In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2 with an amino acid substitution of Ile-36. For example, in some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, wherein amino acid 36 is any amino acid other than isoleucine, for example, amino acid 36 is Gly, Ala , Val, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, wherein amino acid 36 is any amino acid other than isoleucine or valine, for example, amino acid 36 is Gly, Ala Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 36 is Ala, Gly, Leu, or Asp instead of Ile. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 36 is Ala instead of Ile. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 36 is Leu instead of Ile. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 36 is Gly instead of Ile. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 36 is Asp instead of Ile.

  In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 1 with an amino acid substitution of V36. For example, in some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein amino acid 36 is any amino acid other than valine, for example, amino acid 36 is Gly, Ala , Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein amino acid 36 is any amino acid other than isoleucine or valine, for example, amino acid 36 is Gly, Ala Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 1, and amino acid 36 is Ala, Gly, Leu, Glu, or Asp instead of Val. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein amino acid 36 is Glu or Asp instead of Val. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein amino acid 36 is Ala instead of Val. In some instances, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein amino acid 36 is Leu instead of Val. In some instances, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein amino acid 36 is Gly instead of Val. In some instances, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein amino acid 36 is Asp instead of Val. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein amino acid 36 is Glu instead of Val.

  In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2H. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2I.

Q66 Substitution In some cases, a mutant PD-L1 polypeptide of the present disclosure has at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. The amino acid 66 is an amino acid other than glutamine. For example, the amino acid 66 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn. Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 66 is Glu or Asp. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 66 is Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 66 is Asp. In some cases, the mutant PD-L1 polypeptide has an amino acid sequence shown in FIG. 2B (or SEQ ID NO: 2) relative to PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3B). A binding affinity of about 80% to about 100% of the binding affinity exhibited by the containing PD-L1 polypeptide is shown for the PD-1 polypeptide and the wild type B7-1 polypeptide (eg, the amino acid shown in FIG. 3D). Compared to the binding affinity of a wild-type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 3B or SEQ ID NO: 2) to a B7-1 polypeptide comprising the sequence). A decrease in binding affinity for 1 (eg, a decrease in binding affinity for B7-1 of about 40% to about 90%).

  In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B with an amino acid substitution of Q66. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2 having an amino acid substitution of Q48. For example, in some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 66 is any amino acid other than glutamine, eg, amino acid 66 is Gly, Ala, It may be Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 66 is Ala, Gly, Leu, Glu, or Asp instead of Gln. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 66 is Glu or Asp instead of Gln. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 66 is Ala instead of Gln. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 66 is Leu instead of Gln. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 66 is Gly instead of Gln. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 66 is Asp instead of Gln. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 66 is Glu instead of Gln.

  In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2 having an amino acid substitution of Q48. For example, in some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, wherein amino acid 48 is any amino acid other than glutamine, for example, amino acid 48 is Gly, Ala , Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 48 is Ala, Gly, Leu, Glu, or Asp instead of Gln. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 48 is Glu or Asp instead of Gln. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 48 is Ala instead of Gln. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, wherein amino acid 48 is Leu instead of Gln. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 48 is Gly instead of Gln. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 48 is Asp instead of Gln. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 48 is Glu instead of Gln.

  In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2J. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2K.

In some cases, a mutant PD-L1 polypeptide of the present disclosure has at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. The amino acid 72 is an amino acid other than glutamic acid. For example, the amino acid 72 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn. , Gln, Lys, Arg, His, or Asp. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 72 is Arg, Lys, or His. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 72 is Asp, Arg, Lys, or His. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 72 is Arg. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 72 is Lys. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 72 is His. In some cases, a mutant PD-L1 polypeptide of the present disclosure has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B. And amino acid 72 is Asp. In some cases, the mutant PD-L1 polypeptide has an amino acid sequence shown in FIG. 2B (or SEQ ID NO: 2) relative to PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3B). A binding affinity of about 30% to about 60% of the binding affinity exhibited by the containing PD-L1 polypeptide is shown for the PD-1 polypeptide and the wild type B7-1 polypeptide (eg, the amino acid shown in FIG. 3D). Compared to the binding affinity of a wild-type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 3B or SEQ ID NO: 2) to a B7-1 polypeptide comprising the sequence). A decrease in binding affinity for 1 (eg, a decrease in binding affinity for B7-1 of about 40% to about 90%).

  In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B with an E72 amino acid substitution. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2 with an amino acid substitution of E54. For example, in some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 72 is any amino acid other than glutamic acid, for example, amino acid 72 is It may be Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Asp. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 72 is Asp, Arg, His, or Lys instead of Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 72 is Arg, His, or Lys instead of Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 72 is Arg instead of Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 72 is Lys instead of Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 72 is His instead of Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 72 is Asp instead of Glu.

  In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2 with an amino acid substitution of E54. For example, in some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, wherein amino acid 54 is any amino acid other than glutamic acid, for example, amino acid 54 is Gly, Ala , Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Asp. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 54 is Asp, Arg, His, or Lys instead of Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 54 is Arg, His, or Lys instead of Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 54 is Arg instead of Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 54 is Lys instead of Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 54 is His instead of Glu. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 54 is Asp instead of Glu.

  In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2L. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2M.

Fusion polypeptides The present disclosure provides PD-L1 fusion polypeptides. The fusion polypeptides of the present disclosure include a) a mutant PD-L1 polypeptide of the present disclosure, and b) a heterologous fusion partner. In some cases, the heterologous fusion partner is fused to the N-terminus of the mutant PD-L1 polypeptide. In some cases, the heterologous fusion partner is fused to the C-terminus of the mutant PD-L1 polypeptide. In some cases, the PD-L1 fusion polypeptide of the present disclosure is fused to the first heterologous fusion partner fused to the N-terminus of the mutant PD-L1 polypeptide and the C-terminus of the mutant PD-L1 polypeptide. Including a second heterologous fusion partner.

  The full length of a PD-L1 fusion polypeptide of the present disclosure can range from 245 amino acids to 2000 amino acids. For example, the PD-L1 fusion polypeptide of the present disclosure has 245 amino acids to 250 amino acids, 250 amino acids to 275 amino acids, 275 amino acids to 300 amino acids, 300 amino acids to 350 amino acids, 350 amino acids to 350 amino acids, 350 amino acids to 400 amino acids, 400 amino acids to 400 amino acids to 400 amino acids to 450 amino acids, 450 amino acids to 500 amino acids, 500 amino acids to 600 amino acids, 600 amino acids to 700 amino acids, 700 amino acids to 800 amino acids, 800 amino acids to 900 amino acids, 900 amino acids to 1000 amino acids, 1000 amino acids to 1250 amino acids, 1250 It can range from amino acids to 1500 amino acids, 1500 amino acids to 1750 amino acids, or 1750 amino acids to 2000 amino acids.

  Suitable fusion partners include, but are not limited to, a transmembrane domain, an immunoglobulin Fc region (eg, an Fc region of IgG), an antigen binding region of an antibody, a cytokine, an immunoregulatory domain, an intracellular signaling domain, and the like.

T Cell Regulatory Multimeric Polypeptides The present disclosure provides multimeric (eg, heterodimers, heterotrimers) polypeptides. A multimeric polypeptide is a T cell regulatory polypeptide, also referred to herein as “T cell regulatory multimeric polypeptide” or “synTac” (“immunological synapse for T cell activation”). . 1A-1D show schematic diagrams of various T cell regulatory multimeric polypeptides of the present disclosure. The T cell regulatory multimeric polypeptides of the present disclosure are also referred to as “synTac polypeptides” or “multimeric polypeptides”. Where a T cell regulatory multimeric polypeptide of the present disclosure comprises a PD-L1 immunomodulatory polypeptide (eg, a mutant PD-L1 immunomodulatory polypeptide of the present disclosure), such T cell regulatory multimeric polypeptide is Also referred to in the specification as “PD-L1 / synTac”.

  In some cases, a synTac polypeptide of the present disclosure comprises a mutant PD-L1 immunoregulatory polypeptide of the present disclosure. In some cases, a synTac polypeptide of the present disclosure comprises a mutant PD-L1 immunoregulatory polypeptide comprising one amino acid substitution as shown in FIG. 10 or FIG. Thus, in some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure may have a substitution of D26 in the amino acid sequence shown in FIG. 2B or a substitution of D8 in the amino acid sequence shown in SEQ ID NO: 2. Including. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of T37 in the amino acid sequence shown in FIG. 2B or a substitution of T19 in the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of D49 of the amino acid sequence shown in FIG. 2B or a substitution of D31 of the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of L53 of the amino acid sequence shown in FIG. 2B or a substitution of L35 of the amino acid sequence shown in SEQ ID NO: 2. In some cases, the mutant PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure is a substitution of I54 (V54 in mouse PD-L1) of the amino acid sequence shown in FIG. 2B, or the amino acid shown in SEQ ID NO: 2. Contains substitution of I36 in the sequence. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of Y56 in the amino acid sequence shown in FIG. 2B or a substitution of Y38 in the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of Y56 in the amino acid sequence shown in FIG. 2B or a substitution of Y38 in the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of Q66 in the amino acid sequence shown in FIG. 2B or a substitution of Q48 in the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of Q66 in the amino acid sequence shown in FIG. 2B or a substitution of Q48 in the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of E72 of the amino acid sequence shown in FIG. 2B or a substitution of E54 of the amino acid sequence shown in SEQ ID NO: 2. In some cases, the mutant PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure is a substitution of M115 (I115 of mouse PD-L1) of the amino acid sequence shown in FIG. 2B, or the amino acid shown in SEQ ID NO: 2. Includes substitution of M97 in the sequence. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of I116 of the amino acid sequence shown in FIG. 2B, or a substitution of I98 of the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of G119 in the amino acid sequence shown in FIG. 2B or a substitution of G101 in the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of G120 in the amino acid sequence shown in FIG. 2B or a substitution of G102 in the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of G120 in the amino acid sequence shown in FIG. 2B or a substitution of G102 in the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of A121 of the amino acid sequence shown in FIG. 2B or a substitution of A103 of the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of D122 of the amino acid sequence shown in FIG. 2B or a substitution of D104 of the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of Y123 in the amino acid sequence shown in FIG. 2B or a substitution of Y105 in the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of K124 in the amino acid sequence shown in FIG. 2B or a substitution of K106 in the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of R125 of the amino acid sequence shown in FIG. 2B or a substitution of K107 of the amino acid sequence shown in SEQ ID NO: 2.

  As noted above, in some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure has a reduced binding affinity for PD1 compared to the binding affinity of wild-type PD-L1 for PD1. Indicates. In some cases, a multimeric polypeptide of the present disclosure comprising a mutant PD-L1 polypeptide of the present disclosure may also be a wild-type PD-L1 (eg, a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2A or FIG. 2B). Or a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2), as compared to a control multimeric polypeptide comprising a reduced binding affinity for PD1.

  In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure binds B7-1 with a lower affinity compared to the binding affinity of wild-type PD-L1 for B7-1. In some cases, a multimeric polypeptide of the present disclosure comprising a mutant PD-L1 polypeptide of the present disclosure may also be a wild-type PD-L1 (eg, a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2A or FIG. 2B). Or a reduced binding affinity for B7-1 as compared to a control multimeric polypeptide comprising a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

  In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure binds to PD-1 with substantially the same affinity as the binding affinity of wild-type PD-L1 to PD-1. , Binds to B7-1 with lower affinity compared to wild-type PD-L1 binding to B7-1. In some cases, a multimeric polypeptide of the present disclosure comprising a mutant PD-L1 polypeptide of the present disclosure may also be a wild type PD-L1 polypeptide (eg, a PD-L1 comprising the amino acid sequence shown in FIG. 2A or FIG. 2B). Exhibit substantially the same affinity for PD-1 as a control multimeric polypeptide comprising a polypeptide, or a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2; Or a wild-type PD-L1 polypeptide (for example, a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2A or 2B, or a PD-L1 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2). ) Bind to B7-1 with lower binding affinity for B7-1 compared to a control multimeric polypeptide comprising

  In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure binds to PD-1 with a lower affinity compared to binding of wild-type PD-L1 to PD1, and to B7-1 It binds to B7-1 with lower affinity compared to wild type PD-L1 binding. In some cases, a multimeric polypeptide of the present disclosure comprising a mutant PD-L1 polypeptide of the present disclosure may also be a wild-type PD-L1 (eg, a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2A or FIG. 2B). Or a reduced binding affinity for B7-1 as compared to a control multimeric polypeptide comprising a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2), and likewise, A wild-type PD-L1 polypeptide (for example, a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2A or FIG. 2B, or a PD-L1 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2) It binds to B7-1 with lower binding affinity for B7-1 compared to a control multimeric polypeptide comprising.

  In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure binds to PD-1 with a lower affinity compared to binding of wild-type PD-L1 to PD-1, and B7- Binds to B7-1 with substantially the same affinity as the binding affinity of wild type PD-L1 for 1. In some cases, a multimeric polypeptide of the present disclosure comprising a mutant PD-L1 polypeptide of the present disclosure may also be a wild-type PD-L1 (eg, a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2A or FIG. 2B). Or a reduced binding affinity for B7-1 as compared to a control multimeric polypeptide comprising a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2), and likewise, A wild-type PD-L1 polypeptide (for example, a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2A or FIG. 2B, or a PD-L1 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2) A substantially identical affinity to B7-1 is shown for the containing control multimeric polypeptide.

  In some cases, a synTac polypeptide of the present disclosure has a binding affinity for PD1 of a control synTac polypeptide comprising a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2. In comparison, it shows a decrease in binding affinity for PD1. For example, in some cases, a synTac polypeptide of the present disclosure has a binding affinity of a control synTac polypeptide comprising a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2A versus a PD1 polypeptide comprising the amino acid sequence shown in FIG. 3A. It binds to PD1 with a lower binding affinity compared to sex. For example, in some cases, a synTac polypeptide of the present disclosure comprises a control synTac comprising a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2A relative to PD1 (eg, a PD1 polypeptide comprising the amino acid sequence shown in FIG. 3A). At least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% lower, at least 55% compared to the binding affinity of the polypeptide % Low, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or greater than 95% binding affinity It binds to PD1 with sex. As another example, in some cases, a synTac polypeptide of the present disclosure comprises a control synTac polypeptide comprising a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2B versus a PD1 polypeptide comprising the amino acid sequence shown in FIG. 3B. It binds to PD1 with a lower binding affinity compared to the binding affinity. For example, in some cases, a synTac polypeptide of the present disclosure comprises a control synTac comprising a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2B relative to PD1 (eg, a PD1 polypeptide comprising the amino acid sequence shown in FIG. 3B). At least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% lower, at least 55% compared to the binding affinity of the polypeptide % Low, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or greater than 95% binding affinity It binds to PD1 with sex.

  In some cases, a synTac polypeptide of the present disclosure has a binding affinity for PD1 as compared to the binding affinity for PD1 of a control synTac polypeptide comprising a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1. Indicates a decline. For example, in some cases, a synTac polypeptide of the present disclosure is a binding of a control synTac polypeptide comprising a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 against a PD1 polypeptide comprising the amino acid sequence set forth in FIG. 3A. It binds to PD1 with lower binding affinity compared to affinity. For example, in some cases, a synTac polypeptide of the present disclosure includes a PD-L1 polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1 relative to PD1 (eg, a PD1 polypeptide comprising the amino acid sequence set forth in FIG. 3A). at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% lower than at least the binding affinity of the synTac polypeptide, 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or greater than 95% lower Binds to PD1 with affinity

  In some cases, a synTac polypeptide of the present disclosure has a binding affinity for PD1 as compared to the binding affinity for PD1 of a control synTac polypeptide comprising a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2. Indicates a decline. For example, in some cases, a synTac polypeptide of the present disclosure binds a control synTac polypeptide comprising a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 relative to a PD1 polypeptide comprising the amino acid sequence set forth in FIG. 3B. It binds to PD1 with lower binding affinity compared to affinity. For example, in some cases, a synTac polypeptide of the present disclosure includes a PD-L1 polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 2 relative to PD1 (eg, a PD1 polypeptide comprising the amino acid sequence set forth in FIG. 3B). at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% lower than at least the binding affinity of the synTac polypeptide, 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or greater than 95% lower Binds to PD1 with affinity

  In some cases, a synTac polypeptide of the present disclosure has a binding affinity for B7-1 of a control synTac polypeptide comprising a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2. Compared to sex, it shows a decrease in binding affinity for B7-1. For example, in some cases, a synTac polypeptide of the present disclosure can be obtained from a control synTac polypeptide comprising a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2A versus a B7-1 polypeptide comprising the amino acid sequence shown in FIG. 3C. It binds to B7-1 with a lower binding affinity compared to the binding affinity. For example, in some cases, a synTac polypeptide of the present disclosure is a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2A relative to B7-1 (eg, a B7-1 polypeptide comprising the amino acid sequence shown in FIG. 3C). At least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% as compared to the binding affinity of a control synTac polypeptide comprising Low, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or 95% Binds to B7-1 with very low binding affinity . As another example, in some cases, a synTac polypeptide of the present disclosure comprises a control synTac polypeptide comprising a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2B versus a PD1 polypeptide comprising the amino acid sequence shown in FIG. 3D. It binds to B7-1 with a lower binding affinity compared to the binding affinity. For example, in some cases, a synTac polypeptide of the present disclosure is a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2B relative to B7-1 (eg, a B7-1 polypeptide comprising the amino acid sequence shown in FIG. 3D). At least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% as compared to the binding affinity of a control synTac polypeptide comprising Low, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or 95% Binds to B7-1 with very low binding affinity .

  In some cases, a synTac polypeptide of the present disclosure is directed against B7-1 as compared to the binding affinity for B7-1 of a control synTac polypeptide comprising a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1. Shows reduced binding affinity. For example, in some cases, a synTac polypeptide of the present disclosure comprises a control synTac polypeptide comprising a PD-L1 polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1 relative to a B7-1 polypeptide comprising the amino acid sequence set forth in FIG. 3C. It binds to B7-1 with a lower binding affinity compared to the binding affinity. For example, in some cases, a synTac polypeptide of the present disclosure can be a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 for B7-1 (eg, a B7-1 polypeptide comprising the amino acid sequence shown in FIG. 3C). At least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50 compared to the binding affinity of a control synTac polypeptide comprising the peptide % Lower, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or 95 Binds to B7-1 with very low binding affinity To.

  In some cases, a synTac polypeptide of the present disclosure is directed against B7-1 as compared to the binding affinity for B7-1 of a control synTac polypeptide comprising a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2. Shows reduced binding affinity. For example, in some cases, a synTac polypeptide of the present disclosure comprises a control synTac polypeptide comprising a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 relative to a B7-1 polypeptide comprising the amino acid sequence shown in FIG. 3D. It binds to B7-1 with a lower binding affinity compared to the binding affinity. For example, in some cases, a synTac polypeptide of the present disclosure can be a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 relative to B7-1 (eg, a B7-1 polypeptide comprising the amino acid sequence shown in FIG. 3D). At least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50 compared to the binding affinity of a control synTac polypeptide comprising the peptide % Lower, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or 95 Binds to B7-1 with very low binding affinity To.

  As noted above, in some cases, a multimeric polypeptide of the present disclosure, including a mutant PD-L1 polypeptide of the present disclosure, may be a wild type PD-L1 polypeptide (eg, the amino acid sequence shown in FIG. 2A or FIG. 2B). B7-1 (e.g., a PD-L1 polypeptide containing PD, or a PD-L1 polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2) having substantially the same affinity as a control multimeric polypeptide B7-1 polypeptide comprising the amino acid sequence shown in FIG. 3C or FIG. 3D). For example, in some cases, a multimeric polypeptide of the present disclosure comprising a mutant PD-L1 polypeptide of the present disclosure can be a wild-type PD-L1 polypeptide (eg, a PD- comprising the amino acid sequence shown in FIG. 2A or FIG. 2B). At least 75%, at least 80%, at least 85%, at least 90% of the affinity of a control multimeric polypeptide comprising a L1 polypeptide or a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. %, At least 95%, at least 98%, or at least 99% affinity is shown for B7-1 (eg, a B7-1 polypeptide comprising the amino acid sequence shown in FIG. 3C or FIG. 3D).

  As noted above, in some cases, a multimeric polypeptide of the present disclosure, including a mutant PD-L1 polypeptide of the present disclosure, may be a wild type PD-L1 polypeptide (eg, the amino acid sequence shown in FIG. 2A or FIG. 2B). PD1 (eg, FIG. 3A) having substantially the same affinity as a control multimeric polypeptide comprising a PD-L1 polypeptide comprising, or a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. Or PD1 polypeptide comprising the amino acid sequence shown in FIG. 3B). For example, in some cases, a multimeric polypeptide of the present disclosure comprising a mutant PD-L1 polypeptide of the present disclosure can be a wild-type PD-L1 polypeptide (eg, a PD- comprising the amino acid sequence shown in FIG. 2A or FIG. 2B). At least 75%, at least 80%, at least 85%, at least 90% of the affinity of a control multimeric polypeptide comprising a L1 polypeptide or a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. Affinities of%, at least 95%, at least 98%, or at least 99% are shown for PD1 (eg, a PD1 polypeptide comprising the amino acid sequence shown in FIG. 3A or FIG. 3B).

  In some cases, SynTac polypeptides of the present disclosure have a binding affinity for PD1 of 1 nM to about 1 mM. In some cases, synTac polypeptides of the present disclosure have a binding affinity for PD1 of 100 nM to about 100 μM. In some cases, SynTac polypeptides of the present disclosure have a binding affinity for PD1 of about 100 nM to 500 nM. For example, in some cases, a SynTac polypeptide of the present disclosure has about 100 nM to about 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, About 400 nM to about 450 nM, or about 450 nM to about 500 nM of binding affinity for PD1 (eg, a PD1 polypeptide comprising the amino acid sequence shown in FIG. 3A or FIG. 3B). In some cases, SynTac polypeptides of the present disclosure have a binding affinity for PD1 (eg, a PD1 polypeptide comprising the amino acid sequence shown in FIG. 3A or FIG. 3B) of about 500 nM to 1 μM. For example, in some cases, a SynTac polypeptide of the present disclosure has about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, or about 900 nM to about 1 μM PD1 (eg, It has a binding affinity for a PD1 polypeptide comprising the amino acid sequence shown in FIG. 3A or FIG. 3B. In some cases, SynTac polypeptides of the present disclosure have a binding affinity for PD1 (eg, a PD1 polypeptide comprising the amino acid sequence shown in FIG. 3A or FIG. 3B) of about 1 μM to 10 μM. For example, in some cases, a SynTac polypeptide of the present disclosure has about 1 μM to 2 μM, about 2 μM to about 3 μM, about 3 μM to about 4 μM, about 4 μM to about 5 μM, about 5 μM to about 6 μM, about 6 μM to about 7 μM, about It has a binding affinity for PD1 (eg, a PD1 polypeptide comprising the amino acid sequence shown in FIG. 3A or FIG. 3B) of 7 μM to about 8 μM, about 8 μM to about 9 μM, or about 9 μM to about 10 μM. In some cases, SynTac polypeptides of the present disclosure have a binding affinity for PD1 (eg, a PD1 polypeptide comprising the amino acid sequence shown in FIG. 3A or FIG. 3B) of about 10 μM to 100 μM. For example, in some cases, a SynTac polypeptide of the present disclosure has about 10 μM to about 20 μM, about 20 μM to about 30 μM, about 30 μM to about 40 μM, about 40 μM to about 50 μM, about 50 μM to about 60 μM, about 60 μM to about 70 μM, About 70 μM to about 80 μM, about 80 μM to about 90 μM, or about 90 μM to about 100 μM of binding affinity for PD1 (eg, a PD1 polypeptide comprising the amino acid sequence shown in FIG. 3A or FIG. 3B).

  A mutant PD-L1 polypeptide present in a SynTac polypeptide of the present disclosure is a PD-L1 polypeptide (eg, a PD- comprising the amino acid sequence set forth in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2). L1 polypeptide) can have a single amino acid substitution. In some cases, the mutant PD-L1 polypeptide present in a SynTac polypeptide of the present disclosure is a wild type PD-L1 polypeptide (eg, the amino acid sequence shown in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2 Containing 2 to 10 amino acid substitutions (including PD-L1 polypeptide). In some cases, the mutant PD-L1 polypeptide present in a SynTac polypeptide of the present disclosure is a wild type PD-L1 polypeptide (eg, the amino acid sequence shown in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2 Including two PD-L1 polypeptides). In some cases, the mutant PD-L1 polypeptide present in a SynTac polypeptide of the present disclosure is a wild type PD-L1 polypeptide (eg, the amino acid sequence shown in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2 Including PD-L1 polypeptide) with three amino acid substitutions. In some cases, the mutant PD-L1 polypeptide present in a SynTac polypeptide of the present disclosure is a wild type PD-L1 polypeptide (eg, the amino acid sequence shown in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2 (Including PD-L1 polypeptide) having four amino acid substitutions. In some cases, the mutant PD-L1 polypeptide present in a SynTac polypeptide of the present disclosure is a wild type PD-L1 polypeptide (eg, the amino acid sequence shown in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2 (Including PD-L1 polypeptide) having 5 amino acid substitutions. In some cases, the mutant PD-L1 polypeptide present in a SynTac polypeptide of the present disclosure is a wild type PD-L1 polypeptide (eg, the amino acid sequence shown in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2 Including 6 PD-L1 polypeptides). In some cases, the mutant PD-L1 polypeptide present in a SynTac polypeptide of the present disclosure is a wild type PD-L1 polypeptide (eg, the amino acid sequence shown in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2 Including PD-L1 polypeptide) with 7 amino acid substitutions. In some cases, the mutant PD-L1 polypeptide present in a SynTac polypeptide of the present disclosure is a wild type PD-L1 polypeptide (eg, the amino acid sequence shown in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2 Including PD-L1 polypeptide) with 8 amino acid substitutions. In some cases, the mutant PD-L1 polypeptide present in a SynTac polypeptide of the present disclosure is a wild type PD-L1 polypeptide (eg, the amino acid sequence shown in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2 (Including PD-L1 polypeptide) having 9 amino acid substitutions. In some cases, the mutant PD-L1 polypeptide present in a SynTac polypeptide of the present disclosure is a wild type PD-L1 polypeptide (eg, the amino acid sequence shown in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2 Containing PD-L1 polypeptide) with 10 amino acid substitutions.

  In some cases, a multimeric polypeptide of the present disclosure comprises a first polypeptide and a second polypeptide, wherein the first polypeptide is amino terminal (N-terminal) to carboxyl terminal (C-terminal). A) an epitope (eg, a T cell epitope), b) a first major histocompatibility complex (MHC) polypeptide, and c) an immunomodulatory polypeptide (eg, a mutant PD-L1 polypeptide of the present disclosure). And the second polypeptide comprises, in order from the N-terminus to the C-terminus, a) a second MHC polypeptide, and b) an immunoglobulin (Ig) Fc polypeptide. In other cases, a multimeric polypeptide of the present disclosure comprises a first polypeptide and a second polypeptide, wherein the first polypeptide is a) an epitope (in order from N-terminus to C-terminus) For example, a T cell epitope), and b) a first MHC polypeptide, wherein the second polypeptide is a) an immunomodulatory polypeptide (eg, mutant PD-L1 of the present disclosure, in order from N-terminal to C-terminal). Polypeptide), b) a second MHC polypeptide, and c) an Ig Fc polypeptide. In some cases, the first and second MHC polypeptides are class I MHC polypeptides, for example, in some cases, the first MHC polypeptide is an MHC class I β2-microglobulin (B2M or β2M) polypeptide. Yes, the second MHC polypeptide is an MHC class I heavy chain (H chain), or the first MHC polypeptide is an MHC class I heavy chain, and the second MHC polypeptide is an MHC class Iβ2M polypeptide is there. In other cases, the first and second MHC polypeptides are class II MHC polypeptides, for example, in some cases, the first MHC polypeptide is an MHC class II α-chain polypeptide and the second The MHC polypeptide is an MHC class II β-chain polypeptide. In other cases, the first polypeptide is an MHC class II β-chain polypeptide and the second MHC polypeptide is an MHC class II α-chain polypeptide. In some cases, a multimeric polypeptide of the present disclosure comprises two or more mutant PD-L1 immunoregulatory polypeptides of the present disclosure. Where a multimeric polypeptide of the present disclosure includes two or more immunomodulatory polypeptides, in some cases, the two or more immunomodulatory polypeptides may be in the same polypeptide chain and in tandem. Where a multimeric polypeptide of the present disclosure includes two or more immunomodulatory polypeptides, in some cases, two or more mutant PD-L1 immunomodulatory polypeptides include amino acid sequences that are identical to one another. Where a multimeric polypeptide of the present disclosure includes two or more mutant PD-L1 immunomodulatory polypeptides, in some cases, the two or more mutant PD-L1 immunomodulatory polypeptides are present in separate polypeptides. In some cases, multimeric polypeptides of the present disclosure are heterodimers. In some cases, a multimeric polypeptide of the present disclosure is a trimeric polypeptide.

  In some cases, a multimeric polypeptide of the present disclosure comprises a) an N-terminal to C-terminal order, i) an epitope and ii) a first polypeptide comprising a first MHC polypeptide, and b) an N-terminal to C-terminal. In terminal order, it comprises i) a second MHC polypeptide, ii) an Ig Fc polypeptide, and iii) a second polypeptide comprising an immunoregulatory domain (eg, a mutant PD-L1 polypeptide of the present disclosure). In some cases, a multimeric polypeptide of the present disclosure comprises a) an N-terminal to C-terminal order, i) an epitope and ii) a first polypeptide comprising a first MHC polypeptide, and b) an N-terminal to C-terminal. In terminal order, i) a second MHC polypeptide, and ii) a second polypeptide comprising an immunoregulatory domain (eg, a mutant PD-L1 polypeptide of the present disclosure). In some cases, a multimeric polypeptide of the present disclosure comprises a) an N-terminal to C-terminal order, i) an epitope and ii) a first polypeptide comprising a first MHC polypeptide, and b) an N-terminal to C-terminal. In terminal order, it includes a second polypeptide comprising i) an immunoregulatory domain (eg, a mutant PD-L1 polypeptide of the present disclosure), and ii) a second MHC polypeptide. In some cases, a multimeric polypeptide of the present disclosure comprises a) from N-terminal to C-terminal, i) an epitope, ii) a first MHC polypeptide, and iii) an immunoregulatory domain (eg, a mutant PD of the present disclosure). -A first polypeptide comprising -L1 polypeptide) and b) in order from N-terminal to C-terminal i) a second polypeptide comprising a second MHC polypeptide. In some cases, when a multimeric polypeptide of the present disclosure includes a non-Ig backbone, the non-Ig backbone is an XTEN peptide, transferrin polypeptide, Fc receptor polypeptide, elastin-like polypeptide, silk-like polypeptide or silk- It is an elastin-like polypeptide.

  In some cases, a multimeric polypeptide of the present disclosure is monovalent. In some cases, multimeric polypeptides of the present disclosure are multivalent. In some instances, a multivalent multimeric polypeptide of the present disclosure comprises an immunoglobulin Fc polypeptide in one of the first or second polypeptides. For example, depending on the Fc polypeptide present in the multimeric polypeptide of the present disclosure, the multimeric polypeptide may be a homodimer, where two molecules of the multimeric polypeptide are in the homodimer. The two molecules of the multimeric polypeptide may be disulfide bonded to each other, for example via an Fc polypeptide present in the two molecules. As another example, a multimeric polypeptide of the present disclosure can include three, four, or five molecules of a multimeric polypeptide, wherein the molecules of the multimeric polypeptide are, for example, in the molecule They may be disulfide bonded to each other via the existing Fc polypeptides.

  In some cases, a multimeric polypeptide of the present disclosure comprises a first comprising a) i) an epitope, ii) a β2M polypeptide, and iii) a mutant PD-L1 polypeptide of the present disclosure in N-terminal to C-terminal order. A polypeptide, b) i) a class I MHC heavy chain, and ii) a second polypeptide comprising Fc polypeptides in N-terminal to C-terminal order. In some cases, a multimeric polypeptide of the present disclosure comprises a) i) an epitope, and ii) a first polypeptide comprising a β2M polypeptide in N-terminal to C-terminal order, and b) i) a mutation of the present disclosure. A PD-L1 polypeptide, ii) a class I MHC heavy chain, and iii) a second polypeptide comprising an Fc polypeptide in N-terminal to C-terminal order. In some cases, a multimeric polypeptide of the present disclosure comprises a) i) an epitope, ii) a β2M polypeptide, iii) a first mutant PD-L1 polypeptide of the present disclosure, iv) a second mutant PD of the present disclosure. A L1 polypeptide, and v) a first polypeptide comprising a third mutant PD-L1 polypeptide of the present disclosure in N-terminal to C-terminal order; b) i) a class I MHC heavy chain, and ii) A second polypeptide comprising an Fc polypeptide in order from the N-terminus to the C-terminus. In some cases, the first mutant PD-L1 polypeptide, the second mutant PD-L1 polypeptide, and the third mutant PD-L1 polypeptide have the same amino acid sequence. In some cases, the first mutant PD-L1 polypeptide, the second mutant PD-L1 polypeptide, and the third mutant PD-L1 polypeptide differ in amino acid sequence. In some cases, a multimeric polypeptide of the present disclosure comprises a) i) an epitope, and ii) a first polypeptide comprising a β2M polypeptide in N-terminal to C-terminal order, and b) i) the first of the present disclosure. One mutant PD-L1 polypeptide, ii) a second mutant PD-L1 polypeptide of the present disclosure, and iii) a third mutant PD-L1 polypeptide of the present disclosure, iv) a class I MHC heavy chain, and v ) A second polypeptide comprising an Fc polypeptide in N-terminal to C-terminal order. In some cases, the first mutant PD-L1 polypeptide, the second mutant PD-L1 polypeptide, and the third mutant PD-L1 polypeptide have the same amino acid sequence. In some cases, the first mutant PD-L1 polypeptide, the second mutant PD-L1 polypeptide, and the third mutant PD-L1 polypeptide differ in amino acid sequence.

Linker The multimeric polypeptide of the present disclosure can be, for example, between an epitope and an MHC polypeptide, between an MHC polypeptide and an immunomodulatory polypeptide, between an MHC polypeptide and an Ig Fc polypeptide, a first mutant PD. A linker peptide inserted between the L1 polypeptide and the second mutant PD-L1 polypeptide or between the second mutant PD-L1 polypeptide and the third mutant PD-L1 polypeptide Can do.

  Suitable linkers (also referred to as “spacers”) can be easily selected and are of several suitable lengths, eg, 4-10 amino acids, 5-9 amino acids, 6-8 amino acids, Alternatively, it may be any one of 1 amino acid to 25 amino acids, 3 amino acids to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids including 7 amino acids to 8 amino acids. Suitable linkers are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23. , 24, or 25 amino acids in length.

Exemplary linkers include glycine polymer (G) _n , glycine-serine polymers (eg, (GS) _n , (GSGGS) _n (SEQ ID NO: 8) and (GGGS) _n (SEQ ID NO: 9), where n is at least 1), glycine-alanine polymers, alanine-serine polymers and other mobile linkers known in the art. Glycine polymers and glycine-serine polymers can be used, and both Gly and Ser are relatively unstructured and can therefore act as neutral tethers between components. Glycine polymers can be used, and glycine has access to significantly more phiphisi space, even than alanine, and is much less restricted than residues with longer side chains (Rev Computational Chem. 11173-142 (1992)). Exemplary linkers include, but are not limited to, GGSG (SEQ ID NO: 10), GGSGG (SEQ ID NO: 11), GSSGSG (SEQ ID NO: 12), GSGGG (SEQ ID NO: 13), GGGSG (SEQ ID NO: 14), GSSSG (SEQ ID NO: 15). An amino acid sequence including these can be included. Exemplary linkers can include, for example, Gly (Ser4) _n where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some cases, the linker comprises an amino acid sequence (GSSSS) _n where n is 4. In some cases, the linker comprises an amino acid sequence (GSSSS) _n where n is 5. Exemplary linkers can include, for example, ((Gly ₄ ) Ser) _n (SEQ ID NO: 45), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. For example, in some cases, the linker comprises the amino acid sequence (GGGGS) _n , where n is 4. In some cases, the linker comprises the amino acid sequence (GGGGS) _n , where n is 5.

  In some cases, the linker polypeptide present in the first polypeptide of the multimeric polypeptide of the present disclosure has a disulfide bond with a cysteine residue present in the second polypeptide of the multimeric polypeptide of the present disclosure. Contains cysteine residues that can be formed. In some cases, for example, a suitable linker comprises an amino acid sequence (SEQ ID NO: 16) as follows:

Epitopes An epitope present in a multimeric polypeptide of the present disclosure can have a length of about 4 amino acids to about 25 amino acids, eg, the epitope is 4 amino acids (aa) to 10aa, 10aa to 15aa, 15aa to It can have a length of 20aa, or 20aa to 25aa. For example, an epitope present in a multimeric polypeptide of the present disclosure is 4 amino acids (aa), 5aa, 6aa, 7aa, 8aa, 9aa, 10aa, 11aa, 12aa, 13aa, 14aa, 15aa, 16aa, 17aa, 18aa, It can have a length of 19aa, 20aa, 21aa, 22aa, 23aa, 24aa, or 25aa. In some cases, an epitope present in a multimeric polypeptide of the present disclosure has a length of 5-10 amino acids, eg, 5aa, 6aa, 7aa, 8aa, 9aa, or 10aa.

An epitope present in a multimeric polypeptide of the present disclosure specifically binds to a T cell, ie, the epitope specifically binds to an epitope specific T cell. Epitope specific T cells bind to an epitope having a reference amino acid sequence, but do not substantially bind to an epitope different from the reference amino acid sequence. For example, an epitope-specific T cell binds to an epitope having a reference amino acid sequence and, if so bound, with a reference amino acid sequence with an affinity of less than 10 ⁻⁶ M, less than 10 ⁻⁵ M, or less than 10 ⁻⁴ M. Bind to different epitopes. Epitope specific T cells can bind to a specific epitope with an affinity of at least 10 ⁻⁷ M, at least 10 ⁻⁸ M, at least 10 ⁻⁹ M or at least 10 ⁻¹⁰ M.

  Suitable epitopes include, but are not limited to, epitopes present on autoimmune related antigens. Autoimmune antigens include, but are not limited to, myelin basic protein (MBP), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), myelin-associated oligodendrocyte basic protein myocardial myosin (myelin-associated oligodendrotic) basic protein cardiac myosin), cell surface protein (OSP), myelin-related glycoprotein (MAG), neurofilament, interferon omega, transglutaminase, aromatic acid carboxylase, 17-hydroxylase, 21-hydroxylase, cardiolipin, pyruvate dehydration Elementary enzyme, β2 glycoprotein I, phosphatidylserine, apoH, annexin A5, LK -1, soluble liver antigen, carbonic anhydrase, gpIIb-IIIa or 1b-IX, type XVII collagen, tissue transglutaminase, gliadin, GD1a, GQ1b, BP-1, BP-2, epidermal transglutaminase, histidine-tRNA, signal Identification peptide, Mi-2, Jo1, glutamate decarboxylase, HSP60, HSP70, HSP90, IGRP, insulin, carboxypeptidase H, insulinoma antigen-2, IA-2 beta, ICA69, ZnT8, chromogranin A, IAPP, sc170, topoisomerase , Histone, basement membrane collagen type IV, enolase, thyroid peroxidase, thyroglobulin, complement third component, voltage-gated calcium channel, Q-type calcium channel, synaptogagmin synaptogamin), muscarinic acetylcholine receptor M1, SMA, LKM-1, LKM-2, LKM-3, soluble liver antigen, SLA, LP, major peripheral myelin protein P0 (myperipheral myelin protein P0), myeloperoxidase, GQ1b, U1-RNP, Kir4.1, nicotinic acetylcholine receptor, MuSK protein, hypocretin, orexin, keratin, AQP4, Yo, Hu, glutamate receptor, desmoglein 3, p62, sp100, Ro, LA, glycoprotein IIb-IIIa Or Ib-IX, ADAMTS13, cardiolipin, β2 glycoprotein I, HPA-1a, HPA-5b, IFN-gamma, IL-1, TNF-alpha, and GMC F, and the like. Autoimmune antigens also include autoantigens associated with type 1 diabetes, multiple sclerosis, or systemic lupus erythematosus. Islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP) peptide (known as IGRP 206-214), a pancreatic beta cell antigen, may be used as an autoimmune epitope, for example in the context of type 1 diabetes it can. The amino acid sequence of IGRP 206-214 is VYLKTNVFL (SEQ ID NO: 43) (eg, Krishnamurty et al. (2008) J. Immunol. 180: 4458, and Han et al. (2005) J. Clin. Invest. 115: 1879). Other suitable IGRP peptides are described, for example, in Jarchum et al. (2008) Clin. Immunol. 127: 359. Suitable autoantigen epitopes in the context of type 1 diabetes include peptide epitopes of preproinsulin (eg, ALWGPDPAAA (SEQ ID NO: 44)) (eg, Skowera et al. (2008) J. Clin. Invest. 118). : 3390).

  Examples of autoimmune antigens and related autoimmune disorders include myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG) (in either case, multiple sclerosis (MS) CD44, preproinsulin, proinsulin, insulin, glutamate decarboxylase (GAD65), tyrosine phosphatase-like insulinoma antigen 2 (IA2), zinc transporter ((ZnT8), and heat shock protein 60 (HSP6O) (In each case associated with type I diabetes), interphotoreceptor retinoid binding protein (IRBP) associated with autoimmune uveitis, acetylcholine receptor AchR, and insulin-like growth factor-1 receptor (IGF- 1R) (in both cases heavy (Associated with myasthenia), beta-hemolytic streptococcal M protein associated with rheumatic fever (pseudoautoantigen), macrophage migration inhibitory factor associated with arthritis, Ro / LaRNP complex, alpha-fodrin and beta -Fodrine, pancreatic islet cell autoantigen, poly (ADP) ribose polymerase (PARP), NuMA, NOR-90, Ro60 autoantigen, and p27 antigen (all associated with Sjogren's syndrome), Ro60 autoantigen, low density lipo Sm antigen (B / B ′, D1, D2, D3, E, F, G) of a protein, U-1 small nuclear ribonucleoprotein complex, and RNP ribonucleoprotein (in each case associated with lupus erythematosus ), OxLDL, beta (2) GPI, HSP6O / 65, and oxLDL / beta (2) PI (in each case associated with atherosclerosis), cardiac beta (1) -adrenoceptor associated with idiopathic dilated cardiomyopathy (DCM), histidyl-tRNA synthetase (HisRS) associated with myositis , Topoisomerase I, IL-17, or heat shock proteins associated with scleroderma.

MHC polypeptides As noted above, multimeric polypeptides of the present disclosure include MHC polypeptides. In this disclosure, the term “major histocompatibility complex (MHC) polypeptide” refers to human MHC (also referred to as human leukocyte antigen (HLA)) polypeptide, rodent (eg, mouse, rat, etc.) MHC. Polypeptides and other mammalian species (eg, rabbits (eg, rabbits), non-human primates, dogs (eg, dogs), cats (eg, cats), ungulates (eg, horses, cows, sheep, goats) The term “MHC polypeptide” is intended to include class I MHC polypeptides (eg, β-2 microglobulin and MHC), including MHC polypeptides, etc. Class I heavy chains) and MHC class II polypeptides (eg, MHC class IIα and MHC class IIβ polypeptides) ).

  As noted above, in some embodiments of the multimeric polypeptides of the present disclosure, the first and second MHC polypeptides are class I MHC polypeptides, for example, in some cases, the first MHC polypeptide Is an MHC class I β2-microglobulin (β2M) polypeptide and the second MHC polypeptide is an MHC class I heavy chain (H chain). In other cases, the first and second MHC polypeptides are class II MHC polypeptides, for example, in some cases, the first MHC polypeptide is an MHC class II α-chain polypeptide and the second The MHC polypeptide is an MHC class II β-chain polypeptide. In other cases, the first polypeptide is an MHC class II β-chain polypeptide and the second MHC polypeptide is an MHC class II α-chain polypeptide.

  In some cases, the MHC polypeptide of a multimeric polypeptide of the present disclosure is a human MHC polypeptide, which is also referred to as a “human leukocyte antigen” (“HLA”) polypeptide. In some cases, the MHC polypeptide of a multimeric polypeptide of the present disclosure is a class I HLA polypeptide, such as a β2-microglobulin polypeptide or a class I HLA heavy chain polypeptide. Class I HLA heavy chain polypeptides include HLA-A heavy chain polypeptide, HLA-B heavy chain polypeptide, HLA-C heavy chain polypeptide, HLA-E heavy chain polypeptide, HLA-F heavy chain polypeptide and HLA-G heavy chain polypeptide. In some cases, the MHC polypeptide of a multimeric polypeptide of the present disclosure is a class II HLA polypeptide, eg, a class II HLA α chain or a class II HLA β chain. MHC class II polypeptides include MHC class II DPα and β polypeptides, DMα and β polypeptides, DOAα and β polypeptides, DOBα and β polypeptides, DQα and β polypeptides, and DRα and β polypeptides.

  In one example, the MHC class I heavy chain polypeptide of a multimeric polypeptide of the present disclosure is at least 75% relative to amino acids 25-365 of the amino acid sequence of the human HLA-A heavy chain polypeptide shown in FIG. 5A. , Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity.

  In one example, the MHC class I heavy chain polypeptide of the multimeric polypeptide of the present disclosure has at least 75%, at least 80%, relative to amino acids 25-365 of the amino acid sequence of the following human HLA-A heavy chain amino acid sequence: An amino acid sequence having%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity can be included (SEQ ID NO: 17).

  As another example, the MHC class I heavy chain polypeptide of the multimeric polypeptide of the present disclosure is at least 75% relative to amino acids 25-362 of the amino acid sequence of the human HLA-B heavy chain polypeptide shown in FIG. 5B. , Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity.

  As another example, the MHC class I heavy chain polypeptide of the multimeric polypeptide of the present disclosure is at least 75% relative to amino acids 25-362 of the amino acid sequence of the human HLA-C heavy chain polypeptide shown in FIG. 5C. , Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity.

  As another example, an MHC class I heavy chain polypeptide of a multimeric polypeptide of the present disclosure is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, An amino acid sequence having at least 98%, at least 99% or 100% amino acid sequence identity can be included (SEQ ID NO: 18).

  The multimeric polypeptide β2-microglobulin (β2M) polypeptide of the present disclosure may be a human β2M polypeptide, a non-human primate β2M polypeptide, a mouse β2M polypeptide, or the like. In some cases, the β2M polypeptide is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or less than the β2M amino acid sequence shown in FIG. Includes amino acid sequences with 100% amino acid sequence identity. In some cases, the β2M polypeptide is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% relative to amino acids 21-119 of the β2M amino acid sequence shown in FIG. An amino acid sequence having at least 99% or 100% amino acid sequence identity.

  In some cases, the MHC polypeptide comprises a single amino acid substitution relative to a reference MHC polypeptide (the reference MHC polypeptide may be a wild-type MHC polypeptide), where the single amino acid substitution is Amino acids are replaced with cysteine (Cys) residues. Such cysteine residues are present in the second polypeptide chain of the disclosed multimeric polypeptide when present in the MHC polypeptide of the first polypeptide of the disclosed multimeric polypeptide. Can form disulfide bonds with cysteine residues.

  In some cases, the first MHC polypeptide in the first polypeptide of the multimeric polypeptide of the present disclosure and / or the second MHC polypeptide in the second polypeptide of the multimeric polypeptide of the present disclosure is An amino acid substitution to replace a certain amino acid with a cysteine, wherein the substituted cysteine in the first MHC polypeptide forms a disulfide bond with the cysteine in the second MHC polypeptide, The cysteine in the polypeptide forms a disulfide bond with the substituted cysteine in the second MHC polypeptide, or the substituted cysteine in the first MHC polypeptide is a disulfide bond with the substituted cysteine in the second MHC polypeptide. Form.

  For example, in some cases, one of the following pairs of residues in HLAβ2-microglobulin and HLA class I heavy chain is replaced with cysteine (wherein the residue number is that of the mature polypeptide): . 1) β2M residue 12, HLA class I heavy chain residue 236, 2) β2M residue 12, HLA class I heavy chain residue 237, 3) β2M residue 8, HLA class I heavy chain residue 234, 4) β2M residue 10, HLA class I heavy chain residue 235, 5) β2M residue 24, HLA class I heavy chain residue 236, 6) β2M residue 28, HLA class I heavy chain residue 232, 7) β2M residue Group 98, HLA class I heavy chain residue 192, 8) β2M residue 99, HLA class I heavy chain residue 234, 9) β2M residue 3, HLA class I heavy chain residue 120, 10) β2M residue 31 HLA class I heavy chain residue 96, 11) β2M residue 53, HLA class I heavy chain residue 35, 12) β2M residue 60, HLA class I heavy chain residue 96, 13) β2M residue 60, HLA Class I heavy chain residues 122, 14) β2M residue 63, HLA Class I heavy chain residues 27, 15) β2M residue Arg3, HLA class I heavy chain residue Gly120, 16) β2M residue His31, HLA class I heavy chain residue Gln96, 17) β2M residue Asp53, HLA class I Heavy chain residue Arg35, 18) β2M residue Trp60, HLA class I heavy chain residue Gln96, 19) β2M residue Trp60, HLA class I heavy chain residue Asp122, 20) β2M residue Tyr63, HLA class I heavy chain Residues Tyr27, 21) β2M residue Lys6, HLA class I heavy chain residue Glu232, 22) β2M residue Gln8, HLA class I heavy chain residue Arg234, 23) β2M residue Tyr10, HLA class I heavy chain residue Pro235, 24) β2M residue Ser11, HLA class I heavy chain residue Gln242, 25) β2M residue Asn24 HLA class I heavy chain residue Ala236, 26) β2M residue Ser28, HLA class I heavy chain residue Glu232, 27) β2M residue Asp98, HLA class I heavy chain residue His192, and 28) β2M residue Met99, HLA class I heavy chain residue Arg234. The amino acid numbering of the MHC / HLA class I heavy chain is with respect to the mature MHC / HLA class I heavy chain without the signal peptide. For example, in the amino acid sequence shown in FIG. 5A, including the signal peptide, Gly120 is Gly144, Gln96 is Gln120, and so forth. In some cases, the β2M polypeptide contains an R12C substitution and the HLA class I heavy chain contains an A236C substitution, in such cases, between the Cys-12 of the β2M polypeptide and Cys-236 of the HLA class I heavy chain. A disulfide bond is formed. For example, in some cases, residue 236 of the mature HLA-A amino acid sequence (ie, residue 260 of the amino acid sequence shown in FIG. 5A) is replaced with Cys. In some cases, residue 236 of the mature HLA-B amino acid sequence (ie, residue 260 of the amino acid sequence shown in FIG. 5B) is replaced with Cys. In some cases, residue 236 of the mature HLA-C amino acid sequence (ie, residue 260 of the amino acid sequence shown in FIG. 5C) is replaced with Cys. In some cases, residue 32 (corresponding to mature β2M Arg-12) of the amino acid sequence shown in FIG. 6 is replaced with Cys.

  In some cases, the β2M polypeptide comprises the following amino acid sequence:

  In some cases, the β2M polypeptide comprises the following amino acid sequence:

  In some cases, the HLA class I heavy chain polypeptide comprises the following amino acid sequence (SEQ ID NO: 19).

  In some cases, the HLA class I heavy chain polypeptide comprises the following amino acid sequence (SEQ ID NO: 20).

  In some cases, the β2M polypeptide comprises the following amino acid sequence (SEQ ID NO: 42).

  The multimeric polypeptide HLA class I heavy chain polypeptide of the present disclosure comprises the following amino acid sequence (SEQ ID NO: 21). Here, the underlined and bold Cys residues form disulfide bonds with each other in the multimeric polypeptide.

Immunomodulating polypeptides Multimeric polypeptides of the present disclosure include mutant PD-L1 polypeptides as described above. Accordingly, a multimeric polypeptide of the present disclosure includes a mutant PD-L1 polypeptide present in the first polypeptide or the second polypeptide of the multimeric polypeptide of the present disclosure.

Substitution of D26 In some cases, the mutant PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85% relative to the amino acid sequence shown in FIG. 2B , At least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 26 is an amino acid other than aspartic acid, eg, amino acid 26 is Gly, Ala , Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu. In some cases, the mutant PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure is at least 90%, at least 95%, at least 98%, or at least 99% amino acids relative to the amino acid sequence shown in FIG. 2B. Including an amino acid sequence having sequence identity, amino acid 26 is Ala, Gly, Val, Leu, or Ile. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 26 is Ala, Gly, Val, Leu, Ile, or Arg, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide of the present disclosure has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 95% relative to the amino acid sequence shown in FIG. 2B. Amino acid 26 is Ala, including an amino acid sequence having 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 26 is Gly, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 26 is Val, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 26 is Leu, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 26 is Ile, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 26 is Arg, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B (or SEQ ID NO: 2) for PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3B). A wild-type B7-1 polypeptide that exhibits a reduction in binding affinity to a PD-1 polypeptide of about 40% to about 60% compared to the binding affinity of a control multimeric polypeptide comprising a PD-L1 polypeptide. A wild type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2B or SEQ ID NO: 2) against (eg, a B7-1 polypeptide comprising the amino acid sequence shown in FIG. 3D) At least 80%, at least 85%, at least 90%, at least 95%, at least 9% of the binding affinity of the control multimeric polypeptide. %, Or it retains at least 99%.

  In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B with a D26 amino acid substitution. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2 having a D8 amino acid substitution. For example, in some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 26 is any amino acid other than aspartic acid, for example, Amino acid 26 can be Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 26 is Ala, Gly, Val, Leu, or Ile instead of Asp. It is. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 26 is replaced with Ala, Gly, Val, Leu, Ile, Or Arg. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 26 is Ala instead of Asp. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 26 is Val instead of Asp. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 26 is Leu instead of Asp. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 26 is Gly instead of Asp. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 26 is Ile instead of Asp. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 26 is Arg instead of Asp.

  In some cases, the mutated PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80% relative to the amino acid sequence set forth in SEQ ID NO: 2 having a D8 amino acid substitution. At least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, ie, amino acid 8 is other than aspartic acid. For example, in some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 8 is any amino acid other than aspartic acid, for example , Amino acid 8 can be Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 8 is Ala, Gly, Val, Leu, or Asp instead of Asp. Ile. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 8 is replaced with Ala, Gly, Val, Leu, Ile instead of Asp. Or Arg. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 8 is Ala instead of Asp. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 8 is Val instead of Asp. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 8 is Leu instead of Asp. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 8 is Gly instead of Asp. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 8 is Ile. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 8 is Arg instead of Asp.

  In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2D. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2E. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2F. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2G.

Substitution of T37 In some cases, the mutant PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85% relative to the amino acid sequence shown in FIG. 2B. , At least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 37 is an amino acid other than threonine, for example, amino acid 37 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 37 is Gly, Ala, Val, Leu, Ile, Arg, Lys, or His, including an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 37 is Arg, Lys, or His, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 37 is Gly, Ala, Val, Leu, or Ile, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, and amino acid 37 is Arg. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. The amino acid sequence is Lys, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 37 is His, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, and amino acid 37 is Gly. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 37 is Ala, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 37 is Val, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 37 is Leu, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 37 is Ile, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a multimeric polypeptide of the present disclosure has an amino acid sequence shown in FIG. 2B (or SEQ ID NO: 2) relative to PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3B). A binding affinity of about 15% to about 35% of the binding affinity exhibited by a control multimeric polypeptide comprising a PD-L1 polypeptide comprising a PD-L1 polypeptide and a wild-type B7-1 polypeptide ( For example, a control comprising a wild type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 3B or SEQ ID NO: 2) against a B7-1 polypeptide comprising the amino acid sequence shown in FIG. 3D) Shows a reduced binding affinity for B7-1 compared to the binding affinity of the multimeric polypeptide (eg, about 70% to about 70% of the binding affinity for B7-1 Exhibit reduced 90%).

  In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B with a T37 amino acid substitution. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2 having a T19 amino acid substitution. For example, in some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, and amino acid 37 is any amino acid other than threonine, for example, an amino acid 37 may be Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 37 is Gly, Ala, Val, Leu, Ile, Arg, His, or Lys. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 37 is Gly, Ala, Val, Leu, or Ile instead of Thr. It is. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, and amino acid 37 is Arg, His, or Lys instead of Thr. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 37 is Arg instead of Thr. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 37 is Lys instead of Thr. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 37 is His instead of Thr. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, and amino acid 37 is Gly instead of Thr. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 37 is Ala instead of Thr. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 37 is Val instead of Thr. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 37 is Leu instead of Thr. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, with amino acid 37 being Ile instead of Thr.

  In some cases, a mutated PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80% relative to the amino acid sequence set forth in SEQ ID NO: 2 having a T19 amino acid substitution. An amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, ie, amino acid 19 is other than threonine. For example, in some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 19 is any amino acid other than threonine, for example, Amino acid 19 can be Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, wherein amino acid 19 is Gly, Ala, Val, Leu, Ile instead of Thr. Arg, His, or Lys. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, wherein amino acid 19 is Gly, Ala, Val, Leu, or Ile. In some cases, a mutated PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in SEQ ID NO: 2, and amino acid 19 is Arg, His, or Lys instead of Thr. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 19 is Arg instead of Thr. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 19 is Lys instead of Thr. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 19 is His instead of Thr. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 19 is Gly instead of Thr. In some cases, the mutant PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 19 is Ala instead of Thr. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 19 is Val instead of Thr. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 19 is Leu instead of Thr. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 19 is Ile instead of Thr.

I54 Substitution In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85% relative to the amino acid sequence shown in FIG. 2B. , At least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 54 is an amino acid other than isoleucine, for example, amino acid 54 is Gly, Ala, Val, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. %, At least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 54 is an amino acid other than isoleucine or valine, eg, amino acid 54 is Gly, Ala, Leu , Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 54 is Ala, Gly, Leu, Glu, Arg, or Asp, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, and amino acid 54 is Glu or Asp. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, and amino acid 54 is Ala. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, and amino acid 54 is Gly. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, and amino acid 54 is Leu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 54 is Asp, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 54 is Glu, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 54 is Arg, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a multimeric polypeptide of the present disclosure has an amino acid sequence shown in FIG. 2B (or SEQ ID NO: 2) relative to PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 2B). A binding affinity of about 70% to about 100% of the binding affinity exhibited by a control multimeric polypeptide comprising a PD-L1 polypeptide comprising a PD-L1 polypeptide and a wild-type B7-1 polypeptide ( For example, a control comprising a wild type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 3B or SEQ ID NO: 2) against a B7-1 polypeptide comprising the amino acid sequence shown in FIG. 3D) Shows a reduced binding affinity for B7-1 compared to the binding affinity of the multimeric polypeptide (eg, about 40% of the binding affinity for B7-1) Exhibit reduced about 90%).

  In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2A. %, At least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 54 is an amino acid other than valine, for example, amino acid 54 is Gly, Ala, Leu, Ile , Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2A. %, At least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 54 is an amino acid other than isoleucine or valine, eg, amino acid 54 is Gly, Ala, Leu , Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2A. Amino acid 54 is Ala, Gly, Leu, Glu, Arg, or Asp, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2A. Comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, and amino acid 54 is Glu or Asp. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2A. Comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, and amino acid 54 is Ala. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2A. Comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, and amino acid 54 is Gly. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2A. Comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, and amino acid 54 is Leu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2A. Amino acid 54 is Asp, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2A. Amino acid 54 is Glu, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2A. Amino acid 54 is Arg, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a multimeric polypeptide of the present disclosure has an amino acid sequence shown in FIG. 2A (or SEQ ID NO: 1) relative to PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3A). A binding affinity of about 70% to about 100% of the binding affinity exhibited by a control multimeric polypeptide comprising a PD-L1 polypeptide comprising a PD-L1 polypeptide and a wild-type B7-1 polypeptide ( For example, a control comprising a wild-type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 2A or SEQ ID NO: 1) against a B7-1 polypeptide comprising the amino acid sequence shown in FIG. 3C) Shows a reduced binding affinity for B7-1 compared to the binding affinity of the multimeric polypeptide (eg, about 40% of the binding affinity for B7-1) Exhibit reduced about 90%).

  In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B with an amino acid substitution of I54. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2 having an amino acid substitution of I36. For example, in some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, and amino acid 54 is any amino acid other than isoleucine, for example, an amino acid 54 can be Gly, Ala, Val, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 54 is any amino acid other than isoleucine or valine, such as the amino acid 54 can be Gly, Ala, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 54 is Ala, Gly, Leu, Arg, or Asp instead of Ile. It is. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, and amino acid 54 is Ala instead of Ile. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 54 is Leu instead of Ile. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, and amino acid 54 is Gly instead of Ile. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 54 is Asp instead of Ile. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, and amino acid 54 is Arg instead of Ile.

  In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2A with an amino acid substitution of V54. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 1 with an amino acid substitution of V36. For example, in some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2A, wherein amino acid 54 is any amino acid other than valine, such as the amino acid 54 can be Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2A, and amino acid 54 is any amino acid other than isoleucine or valine, such as the amino acid 54 can be Gly, Ala, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2A, wherein amino acid 54 is replaced with Ala, Gly, Leu, Glu, Arg, Or Asp. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2A, and amino acid 54 is Glu or Asp instead of Val. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2A, wherein amino acid 54 is Ala instead of Val. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2A, and amino acid 54 is Leu instead of Val. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2A, and amino acid 54 is Gly instead of Val. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2A, wherein amino acid 54 is Asp instead of Val. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2A, and amino acid 54 is Glu instead of Val. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2A, and amino acid 54 is Arg instead of Val.

  In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure has at least 70%, at least 75%, at least Comprises an amino acid sequence having 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, ie, amino acid 36 is other than isoleucine. For example, in some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 36 is any amino acid other than isoleucine, for example, Amino acid 36 can be Gly, Ala, Val, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, the mutant PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, wherein amino acid 36 is any amino acid other than isoleucine or valine, for example, Amino acid 36 can be Gly, Ala, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, wherein amino acid 36 is Ala, Gly, Leu, Arg, or Ile instead of Asp. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 36 is Ala instead of Ile. In some cases, the mutant PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 36 is Leu instead of Ile. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 36 is Gly instead of Ile. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 36 is Asp instead of Ile. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 36 is Arg instead of Ile.

  In some cases, the mutant PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80% relative to the amino acid sequence set forth in SEQ ID NO: 1 with a V36 amino acid substitution. , Amino acid sequences having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. For example, in some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 1, and amino acid 36 is any amino acid other than valine, for example, Amino acid 36 can be Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 1, and amino acid 36 is any amino acid other than isoleucine or valine, such as Amino acid 36 can be Gly, Ala, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein amino acid 36 is Ala, Gly, Leu, Glu, or Asp. In some cases, a mutant PD-L1 polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein amino acid 36 is Glu or Asp instead of Val. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 1, and amino acid 36 is Ala instead of Val. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 1, and amino acid 36 is Leu instead of Val. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 1, and amino acid 36 is Gly instead of Val. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 1, and amino acid 36 is Asp instead of Val. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 1, and amino acid 36 is Glu instead of Val. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 1, and amino acid 36 is Arg instead of Val.

  In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2H. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG.

Q66 Substitution In some cases, the mutant PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85% relative to the amino acid sequence shown in FIG. 2B. , At least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 66 is an amino acid other than glutamine, for example, amino acid 66 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 66 is Glu or Asp, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 66 is Glu, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 66 is Asp, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a multimeric polypeptide of the present disclosure has an amino acid sequence shown in FIG. 2B (or SEQ ID NO: 2) relative to PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3B). A binding affinity of about 80% to about 100% of the binding affinity exhibited by a control multimeric polypeptide comprising a PD-L1 polypeptide comprising a PD-1 polypeptide, wherein the wild type B7-1 polypeptide ( For example, a control comprising a wild type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 3B or SEQ ID NO: 2) against a B7-1 polypeptide comprising the amino acid sequence shown in FIG. 3D) Shows a reduced binding affinity for B7-1 compared to the binding affinity of the multimeric polypeptide (eg, about 40% of the binding affinity for B7-1) Exhibit reduced about 90%).

  In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B with an amino acid substitution of Q66. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2 with a Q48 amino acid substitution. For example, in some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 66 is any amino acid other than glutamine, such as the amino acid 66 may be Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 66 is Ala, Gly, Leu, Glu, or Asp instead of Gln. It is. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 66 is Glu or Asp instead of Gln. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 66 is Ala instead of Gln. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 66 is Leu instead of Gln. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 66 is Gly instead of Gln. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 66 is Asp instead of Gln. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 66 is Glu instead of Gln.

  In some cases, a mutated PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80% relative to the amino acid sequence set forth in SEQ ID NO: 2 having a Q48 amino acid substitution. An amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, ie, amino acid 48 is other than glutamine. For example, in some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, wherein amino acid 48 is any amino acid other than glutamine, for example, Amino acid 48 can be Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, wherein amino acid 48 is Ala, Gly, Leu, Glu, or Gln instead of Asp. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 48 is Glu or Asp instead of Gln. In some cases, the mutant PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 48 is Ala instead of Gln. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 48 is Leu instead of Gln. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 48 is Gly instead of Gln. In some cases, the mutant PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 48 is Asp instead of Gln. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 48 is Glu instead of Gln.

  In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2J. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2K.

E72 Substitution In some cases, the mutant PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85% relative to the amino acid sequence shown in FIG. 2B. , At least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 72 is an amino acid other than glutamic acid, for example, amino acid 72 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Asp. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Comprising amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity; In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Wherein the amino acid 72 is Asp, Arg, Lys, or His. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Comprising amino acid sequence with%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, amino acid 72 is Arg. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Comprising amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, and amino acid 72 is Lys. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 72 is His, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 72 is Asp, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a multimeric polypeptide of the present disclosure has an amino acid sequence shown in FIG. 2B (or SEQ ID NO: 2) relative to PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3B). A binding affinity of about 30% to about 60% of the binding affinity exhibited by a control multimeric polypeptide comprising a PD-L1 polypeptide comprising a PD-L1 polypeptide, wherein the wild-type B7-1 polypeptide ( For example, a control comprising a wild type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 3B or SEQ ID NO: 2) against a B7-1 polypeptide comprising the amino acid sequence shown in FIG. 3D) Shows a reduced binding affinity for B7-1 compared to the binding affinity of the multimeric polypeptide (eg, about 40% to about 40% of the binding affinity for B7-1 Exhibit reduced 90%).

  In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B with an E72 amino acid substitution. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2 with an amino acid substitution of E54. For example, in some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 72 is any amino acid other than glutamic acid, 72 may be Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Asp. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, and amino acid 72 is Asp, Arg, His, or Lys instead of Glu. . In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, and amino acid 72 is Arg, His, or Lys instead of Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 72 is Arg instead of Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 72 is Lys instead of Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 72 is His instead of Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 72 is Asp instead of Glu.

  In some cases, a mutated PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80% relative to the amino acid sequence set forth in SEQ ID NO: 2 having an amino acid substitution of E54. An amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, ie, amino acid 54 is other than glutamic acid. For example, in some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 54 is any amino acid other than glutamic acid, for example, Amino acid 54 can be Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Asp. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 54 is Asp, Arg, His, or Lys instead of Glu. is there. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 54 is Arg, His, or Lys instead of Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 54 is Arg instead of Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 54 is Lys instead of Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 54 is His instead of Glu. In some cases, the mutant PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 54 is Asp instead of Glu.

  In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2L. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2M.

Substitution of Y56 In some cases, the mutant PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85% relative to the amino acid sequence shown in FIG. 2B. , At least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 56 is an amino acid other than tyrosine, for example, amino acid 56 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 56 is Ala, Gly, Val, Leu, or Ile, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 56 is Asp or Glu, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 56 is Arg, His, or Lys, including an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 56 is Ala, Asp, or Arg, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, and amino acid 56 is Arg. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. The amino acid 56 is Asp, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 56 is Ala, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a multimeric polypeptide of the present disclosure has an amino acid sequence shown in FIG. 2B (or SEQ ID NO: 2) relative to PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3B). A binding affinity of about 50% to about 100% of the binding affinity exhibited by a control multimeric polypeptide comprising a PD-L1 polypeptide comprising a PD-1 polypeptide and a wild type B7-1 polypeptide ( For example, a control comprising a wild type PD-L1 polypeptide (eg, a PD-L1 polypeptide comprising the amino acid sequence shown in FIG. 3B or SEQ ID NO: 2) against a B7-1 polypeptide comprising the amino acid sequence shown in FIG. 3D) Shows a reduced binding affinity for B7-1 compared to the binding affinity of the multimeric polypeptide (eg, about 60% of the binding affinity for B7-1) Exhibit reduced about 95%).

  In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B with an amino acid substitution of Y56. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2 having an amino acid substitution of Y38. For example, in some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 56 is any amino acid other than tyrosine, 56 may be Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 56 is Ala, Val, Gly, Leu, or Ile instead of Tyr. It is. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, and amino acid 56 is Arg, His, or Lys instead of Tyr. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, and amino acid 56 is Asp or Glu instead of Tyr. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, with amino acid 56 being Arg instead of Tyr. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, with amino acid 56 being Asp instead of Tyr. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, and amino acid 56 is Ala instead of Tyr.

  In some cases, the mutated PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80% relative to the amino acid sequence set forth in SEQ ID NO: 2 having an amino acid substitution of Y38. An amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, ie, amino acid 38 is other than tyrosine. For example, in some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, wherein amino acid 38 is any amino acid other than tyrosine, Amino acid 38 can be Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, the mutant PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 38 is Arg, His, or Lys instead of Tyr. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 38 is Asp or Glu instead of Tyr. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, wherein amino acid 38 is Ala, Gly, Val, Leu, or Ile. In some cases, the mutant PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 38 is Arg instead of Tyr. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 38 is Ala instead of Tyr. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 38 is Asp instead of Tyr.

Substitution of G119 In some cases, the mutant PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85% relative to the amino acid sequence shown in FIG. 2B. , Amino acid 119 is an amino acid other than glycine, for example, amino acid 119 is Ala, Val, and at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 119 is Ala, Val, Leu, or Ile, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 119 is Asp or Glu. The amino acid sequence has an amino acid sequence identity of%, at least 95%, at least 98%, or at least 99%. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Comprising amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, amino acid 119 is Arg, His, or Lys. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 119 is Asp, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 119 is Arg, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Amino acid 119 is Ala, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a multimeric polypeptide of the present disclosure has an amino acid sequence shown in FIG. 2B (or SEQ ID NO: 2) relative to PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3B). A binding affinity of about 20% to about 50% or about 50% to 100% of the binding affinity exhibited by a control multimeric polypeptide comprising a PD-L1 polypeptide comprising A wild-type PD-L1 polypeptide (eg, PD- containing the amino acid sequence shown in FIG. 3B or SEQ ID NO: 2) against a type B7-1 polypeptide (eg, a B7-1 polypeptide containing the amino acid sequence shown in FIG. 3D) Display reduced binding affinity for B7-1 (eg, for B7-1) as compared to the binding affinity of a control multimeric polypeptide comprising (L1 polypeptide) Exhibit reduced about 60% to about 95% of the binding affinity).

  In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B with the G119 amino acid substitution. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2 having an amino acid substitution of G101. For example, in some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 119 is any amino acid other than glycine, for example, an amino acid 119 can be Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 119 is Ala, Val, Leu, or Ile instead of Gly. . In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 119 is Arg, His, or Lys instead of Gly. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 119 is Asp or Glu instead of Gly. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 119 is Arg instead of Gly. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 119 is Asp instead of Gly. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 119 is Ala instead of Gly.

  In some cases, the mutant PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80% relative to the amino acid sequence set forth in SEQ ID NO: 2 having an amino acid substitution of G101. At least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, ie, amino acid 101 is other than glycine. For example, in some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 101 is any amino acid other than glycine, for example, Amino acid 101 can be Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 101 is Arg, His, or Lys instead of Gly. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 101 is Asp or Glu instead of Gly. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 101 is Ala, Val, Leu, or Ile instead of Gly. is there. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 101 is Arg instead of Gly. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 101 is Ala instead of Gly. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 101 is Asp instead of Gly.

Substitution of G120 In some cases, the mutant PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85% relative to the amino acid sequence shown in FIG. 2B. , At least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 120 is an amino acid other than glycine, eg, amino acid 120 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. The amino acid 120 is Ala, Val, Leu, or Ile, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Wherein the amino acid 120 is Asp or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 120 is Arg, His, or Lys. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. The amino acid 120 is Asp, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. Comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 120 is Arg. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the amino acid sequence shown in FIG. 2B. The amino acid 120 is Ala, comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. In some cases, a multimeric polypeptide of the present disclosure has an amino acid sequence shown in FIG. 2B (or SEQ ID NO: 2) relative to PD-1 (eg, a PD-1 polypeptide comprising the amino acid sequence shown in FIG. 3B). A binding affinity of about 20% to about 50% or about 50% to 100% of the binding affinity exhibited by a control multimeric polypeptide comprising a PD-L1 polypeptide comprising A wild-type PD-L1 polypeptide (eg, PD- containing the amino acid sequence shown in FIG. 3B or SEQ ID NO: 2) against a type B7-1 polypeptide (eg, a B7-1 polypeptide containing the amino acid sequence shown in FIG. 3D) A reduced binding affinity for B7-1 (eg, for B7-1) as compared to the binding affinity of a control multimeric polypeptide comprising (L1 polypeptide) Exhibit reduced about 60% to about 95% of the binding affinity).

  In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B with an amino acid substitution of G120. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2 having an amino acid substitution of G102. For example, in some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 120 is any amino acid other than glycine, such as the amino acid 120 can be Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 120 is Ala, Val, Leu, or Ile instead of Gly. . In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 120 is Arg, His, or Lys instead of Gly. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 120 is Asp or Glu instead of Gly. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 120 is Arg instead of Gly. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 120 is Asp instead of Gly. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in FIG. 2B, wherein amino acid 120 is Ala instead of Gly.

  In some cases, the mutated PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure is at least 70%, at least 75%, at least 80% relative to the amino acid sequence set forth in SEQ ID NO: 2 having an amino acid substitution of G102. An amino acid sequence having an amino acid sequence identity of at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, ie, amino acid 102 is other than glycine. For example, in some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 102 is any amino acid other than glycine, for example, Amino acid 101 can be Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 102 is Arg, His, or Lys instead of Gly. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 102 is Asp or Glu instead of Gly. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 102 is Ala, Val, Leu, or Ile instead of Gly. is there. In some cases, the mutant PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 102 is Arg instead of Gly. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 102 is Ala instead of Gly. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 2, and amino acid 102 is Asp instead of Gly.

Multiple mutant PD-L1 immunomodulatory domains In some cases, a multimeric polypeptide of the present disclosure comprises a single mutant PD-L1 immunomodulatory polypeptide.

  In some cases, a multimeric polypeptide of the present disclosure comprises two mutant PD-L1 immunomodulatory polypeptides. In some cases, two mutant PD-L1 immunoregulatory polypeptides are present in tandem with the polypeptide chain. In some cases, the two mutant PD-L1 immunoregulatory polypeptides are present on separate polypeptide chains. In some cases, the two mutant PD-L1 immunoregulatory polypeptides are present on separate polypeptide chains of the multimeric polypeptide. In some cases, the two mutant PD-L1 polypeptides have amino acid sequences that are identical to each other. In some cases, the two mutant PD-L1 polypeptides have different amino acid sequences (eg, these two differ from each other by at least one amino acid).

  In some cases, a multimeric polypeptide of the present disclosure comprises three mutant PD-L1 immunomodulatory polypeptides. In some cases, the three mutant PD-L1 immunoregulatory polypeptides are present in tandem with the polypeptide chain. In some cases, one of the three mutant PD-L1 immunoregulatory polypeptides is present on a separate polypeptide chain in the multimeric polypeptide from the other two mutant PD-L1 immunomodulatory polypeptides. In some cases, the three mutant PD-L1 polypeptides have amino acid sequences that are identical to one another. In some cases, each of the three mutant PD-L1 polypeptides has a different amino acid sequence (eg, each has at least one amino acid different from the other two).

Skeletal Polypeptides The T cell regulatory multimeric polypeptides of the present disclosure include an Fc polypeptide or another suitable scaffold polypeptide.

  Suitable scaffold polypeptides include antibody-based scaffold polypeptides and non-antibody-based scaffolds. Non-antibody based scaffolds include, for example, albumin, XTEN (extended recombination) polypeptide, transferrin, Fc receptor polypeptide, elastin-like polypeptide (eg, Hassonneh et al. (2012) Methods Enzymol. 502: 215). See, for example, X is a polypeptide containing a pentapeptide repeat unit of any amino acid other than proline (Val-Pro-Gly-X-Gly), albumin binding polypeptide, silk-like polypeptide (eg, Valluzzi et al. (2002) Philos Trans R Soc London B Biol Sci. 357: 165)), silk-elastin-like polypeptides (SELP: eg, Megged et al. (2002) Adv Drug Deliv Rev. 54: 1075). Suitable XTEN polypeptides include, for example, those disclosed in WO2009 / 023270, WO2010 / 091122, WO2007 / 103515, US2010 / 0189682 and US2009 / 0092582, see Schellenberger et al. (2009) Nat Biotechnol. 27: 1186). Suitable albumin polypeptides include, for example, human serum albumin.

  A suitable backbone polypeptide is in some cases a half-life extending polypeptide. Thus, in some cases, a suitable backbone polypeptide extends the in vivo half-life (eg, serum half-life) of the multimeric polypeptide as compared to a control multimeric polypeptide that lacks the backbone polypeptide. For example, in some cases, the backbone polypeptide has an in vivo half-life (eg, serum half-life) of the multimeric polypeptide of at least about 10%, at least about 15 compared to a control multimeric polypeptide that lacks the backbone polypeptide. %, At least about 20%, at least about 25%, at least about 50%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 25 times, at least about 50 times, at least Extend about 100 times or more than 100 times. In one example, in some cases, the Fc polypeptide has an in vivo half-life (eg, serum half-life) of the multimeric polypeptide of at least about 10%, as compared to a control multimeric polypeptide that lacks the Fc polypeptide, At least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 25 times, at least about 50 times Extend at least about 100 times or more than 100 times.

Fc polypeptide In some cases, the first and / or second polypeptide chain of a multimeric polypeptide of the present disclosure comprises an Fc polypeptide. The Fc polypeptide of the multimeric polypeptide of the present disclosure may be human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc and the like. In some cases, the Fc polypeptide is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about the amino acid sequence of the Fc region shown in FIGS. Amino acid sequences having about 95%, at least about 98%, at least about 99% or 100% amino acid sequence identity. In some cases, the Fc region is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% relative to the human IgG1 Fc polypeptide shown in FIG. 4A. An amino acid sequence having at least about 98%, at least about 99% or 100% amino acid sequence identity. In some cases, the Fc region is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% relative to the human IgG1 Fc polypeptide shown in FIG. 4A. An amino acid sequence having at least about 98%, at least about 99% or 100% amino acid sequence identity, and comprising N77 substitutions, eg, an Fc polypeptide comprises an N77A substitution. In some cases, the Fc polypeptide is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95 relative to the human IgG2 Fc polypeptide shown in FIG. 4A. %, At least about 98%, at least about 99% or 100% amino acid sequence identity, eg, the Fc polypeptide is against amino acids 99-325 of the human IgG2 Fc polypeptide shown in FIG. 4A Having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid sequence identity Contains amino acid sequence. In some cases, the Fc polypeptide is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95 relative to the human IgG3 Fc polypeptide shown in FIG. 4A. %, At least about 98%, at least about 99% or 100% amino acid sequence identity, eg, the Fc polypeptide is against amino acids 19-246 of the human IgG3 Fc polypeptide shown in FIG. 4A Having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid sequence identity Contains amino acid sequence. In some cases, the Fc polypeptide is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95 relative to the human IgM Fc polypeptide shown in FIG. 4B. %, At least about 98%, at least about 99% or 100% amino acid sequence identity, eg, the Fc polypeptide is against amino acids 1-276 for the human IgM Fc polypeptide shown in FIG. 4B Having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid sequence identity Contains amino acid sequence. In some cases, the Fc polypeptide is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95 relative to the human IgA Fc polypeptide shown in FIG. 4C. %, At least about 98%, at least about 99%, or 100% amino acid sequence identity, eg, the Fc polypeptide is against amino acids 1-234 relative to the human IgA Fc polypeptide shown in FIG. 4C Having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid sequence identity Contains amino acid sequence.

Additional Polypeptides The polypeptide chain of a multimeric polypeptide of the present disclosure can include one or more polypeptides in addition to those described above. Suitable additional polypeptides include epitope tags and affinity domains. The one or more additional polypeptides may be the N-terminus of the polypeptide chain of the multimeric polypeptide of the present disclosure, the C-terminus of the polypeptide chain of the multimeric polypeptide of the present disclosure or the polypeptide of the multimeric polypeptide of the present disclosure. It can be contained within the chain.

Epitope tag Suitable epitope tags include, but are not limited to, hemagglutinin (HA: eg YPYDVPDYA (SEQ ID NO: 22)), FLAG (eg DYKDDDDK (SEQ ID NO: 23)), c-myc (eg EQKLISEEDL (SEQ ID NO: 24 )).

Affinity Domain An affinity domain comprises a peptide sequence that can interact with a binding partner useful for identification or purification, such as that immobilized on a solid support. DNA sequences encoding multiple consecutive single amino acids, such as histidine, are used for one-step purification of recombinant proteins by high affinity binding to a resin column such as nickel sepharose when fused to the expressed protein be able to. Exemplary affinity domains include His5 (HHHHH) (SEQ ID NO: 25), HisX6 (HHHHHH) (SEQ ID NO: 26), C-myc (EQKLISEEDL) (SEQ ID NO: 27), Flag (DYKDDDDK) (SEQ ID NO: 28), StrepTag (WSHPQFEK) (SEQ ID NO: 29), hemagglutinin, eg, HA tag (YPYDVPDYA) (SEQ ID NO: 30), glutathione-S-transferase (GST), thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO: 31), Phe-His -His-Thr (SEQ ID NO: 32), chitin binding domain, S-peptide, T7 peptide, SH2 domain, C-terminal RNA tag, WEAAARECACCRECARA (SEQ ID NO: 33), metal binding domain, eg zinc Combined domains or calcium binding domains such as calcium binding proteins such as calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, vidinine, VILIP, neurocalcin, hypocalcin, friquenin, caltractin, calpain large Examples include subunits, S100 protein, parvalbumin, calbindin D9K, calbindin D28K and those derived from calretinin, intein, biotin, streptavidin, MyoD, Id, leucine zipper sequences and maltose binding protein.

Exemplary Multimeric Polypeptides Exemplary multimeric polypeptides of the present disclosure are described below.

D26 Substitution In some cases, a multimeric polypeptide of the present disclosure comprises a) i) an epitope, ii) a β2M polypeptide, and iii) at least 70%, at least 75%, at least relative to the amino acid sequence shown in FIG. A variant PD-L1 polypeptide comprising an amino acid sequence having 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 26 is aspartic acid For example, amino acid 26 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu. For example, amino acid 26 is Ala or A a mutant PD-L1 polypeptide that is g, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, relative to the amino acid sequence set forth in SEQ ID NO: 2. Or a mutant PD-L1 polypeptide comprising an amino acid sequence having at least 99% amino acid sequence identity, wherein amino acid 8 is an amino acid other than aspartic acid, for example, amino acid 8 is Gly, Ala, Val, Leu , Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu, for example, a mutant PD-L1 poly in which amino acid 8 is Ala or Arg A first poly comprising peptides in the order N-terminal to C-terminal Comprising a peptide, b) i) and a second polypeptide Class I MHC heavy chain, and ii) an Fc polypeptide from the N-terminus comprising in the order of C-terminal, the. In some cases, a multimeric polypeptide of the present disclosure comprises a) i) an epitope, and ii) a first polypeptide comprising a β2M polypeptide in N-terminal to C-terminal order, and b) i) shown in FIG. 2B. A mutation comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity PD-L1 polypeptide, wherein amino acid 26 is an amino acid other than aspartic acid, for example, amino acid 26 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys , Met, Asn, Gln, Lys, Arg, His, or Glu, eg For example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% of the mutant PD-L1 polypeptide in which amino acid 26 is Ala or Arg, or the amino acid sequence shown in SEQ ID NO: 2, A mutant PD-L1 polypeptide comprising an amino acid sequence having at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 8 is an amino acid other than aspartic acid, eg, amino acid 8 is , Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu, for example, amino acid 8 is Ala or A mutant PD-L1 polypeptide that is Arg; And iii) a second polypeptide comprising an Fc polypeptide in N-terminal to C-terminal order. In some cases, a multimeric polypeptide of the present disclosure comprises a) i) an epitope, ii) a β2M polypeptide, iii) a first mutant PD-L1 polypeptide of the present disclosure, iv) a second mutant PD of the present disclosure. A L1 polypeptide, and v) a first polypeptide comprising a third variant PD-L1 polypeptide of the present disclosure in N-terminal to C-terminal order; b) i) a class I MHC heavy chain, and ii) A second polypeptide comprising an Fc polypeptide in order from the N-terminus to the C-terminus. In some cases, each of the first mutant PD-L1 polypeptide, the second mutant PD-L1 polypeptide, and the third mutant PD-L1 polypeptide is i) at least relative to the amino acid sequence shown in FIG. 2B. An amino acid sequence having 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 26 is aspartic acid For example, amino acid 26 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu. For example, an amino acid 26 is Ala or Arg. Or ii) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or with respect to the amino acid sequence shown in SEQ ID NO: 2, or An amino acid sequence having at least 99% amino acid sequence identity, wherein amino acid 8 is an amino acid other than aspartic acid, for example, amino acid 8 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu. For example, amino acid 8 includes an amino acid sequence that is Ala or Arg. In some cases, a multimeric polypeptide of the present disclosure comprises a) i) an epitope, and ii) a first polypeptide comprising a β2M polypeptide in N-terminal to C-terminal order, and b) i) the first of the present disclosure. One mutant PD-L1 polypeptide, ii) a second mutant PD-L1 polypeptide of the present disclosure, iii) a third mutant PD-L1 polypeptide of the present disclosure, iv) a class I MHC heavy chain, and v) A second polypeptide comprising an Fc polypeptide in order from the N-terminus to the C-terminus. In some cases, each of the first mutant PD-L1 polypeptide, the second mutant PD-L1 polypeptide, and the third mutant PD-L1 polypeptide is i) at least relative to the amino acid sequence shown in FIG. 2B. An amino acid sequence having 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 26 is aspartic acid For example, amino acid 26 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu. For example, an amino acid 26 is Ala or Arg. Or ii) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or with respect to the amino acid sequence shown in SEQ ID NO: 2, or An amino acid sequence having at least 99% amino acid sequence identity, wherein amino acid 8 is an amino acid other than aspartic acid, for example, amino acid 8 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu. For example, amino acid 8 includes an amino acid sequence that is Ala or Arg. In some cases, a multimeric polypeptide of the present disclosure comprises a) i) an epitope, and ii) a first polypeptide comprising a β2M polypeptide in N-terminal to C-terminal order, and b) i) the first of the present disclosure. 1 mutant PD-L1 polypeptide, ii) linker, iii) second mutant PD-L1 polypeptide of the present disclosure, iv) linker, v) third mutant PD-L1 polypeptide of the present disclosure, vi) class I MHC heavy chain, and vii) a second polypeptide comprising an Fc polypeptide in N-terminal to C-terminal order. In some cases, each of the first mutant PD-L1 polypeptide, the second mutant PD-L1 polypeptide, and the third mutant PD-L1 polypeptide is i) at least relative to the amino acid sequence shown in FIG. 2B. An amino acid sequence having 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 26 is aspartic acid For example, amino acid 26 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu. For example, an amino acid 26 is Ala or Arg. Or ii) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or with respect to the amino acid sequence shown in SEQ ID NO: 2, or An amino acid sequence having at least 99% amino acid sequence identity, wherein amino acid 8 is an amino acid other than aspartic acid, for example, amino acid 8 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu. For example, amino acid 8 includes an amino acid sequence that is Ala or Arg. In some cases, the linker comprises the sequence (GSSSS) n, where n is 1, 2, 3, 4, or 5. In some cases, n is 4. In some cases, n is 5.

Substitution of T37 In some cases, a multimeric polypeptide of the present disclosure comprises a) i) an epitope, ii) a β2M polypeptide, and iii) at least 70%, at least 75%, at least relative to the amino acid sequence shown in FIG. A mutant PD-L1 polypeptide comprising an amino acid sequence having 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 37 is other than threonine For example, amino acid 37 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. For example, amino acid 37 is Gly, Ala, Va A mutated PD-L1 polypeptide that is Leu, Ile, Arg, Lys, or His, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90 with respect to the amino acid sequence set forth in SEQ ID NO: 2. A variant PD-L1 polypeptide comprising an amino acid sequence having%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 19 is any amino acid other than threonine; Amino acid 19 can be Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu, for example, amino acid 19 But Gly, Ala, Val, Leu, I a first polypeptide comprising a mutant PD-L1 polypeptide that is e, Arg, Lys, or His in N-terminal to C-terminal order; b) i) a class I MHC heavy chain, and ii) an Fc polypeptide. A second polypeptide comprising an N-terminal to C-terminal order. In some cases, a multimeric polypeptide of the present disclosure comprises a) i) an epitope, and ii) a first polypeptide comprising a β2M polypeptide in N-terminal to C-terminal order, and b) i) shown in FIG. 2B. A mutation comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity PD-L1 polypeptide, wherein amino acid 37 is an amino acid other than threonine, for example, amino acid 37 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu, for example A mutant PD-L1 polypeptide in which amino acid 37 is Gly, Ala, Val, Leu, Ile, Arg, Lys, or His, or at least 70%, at least 75%, at least relative to the amino acid sequence shown in SEQ ID NO: 2 A mutant PD-L1 polypeptide comprising an amino acid sequence having 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 19 is other than threonine For example, amino acid 19 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or For example, amino acid 19 is A mutated PD-L1 polypeptide that is Gly, Ala, Val, Leu, Ile, Arg, Lys, or His, and ii) a second polypeptide comprising an Fc polypeptide in N-terminal to C-terminal order. Including. In some cases, a multimeric polypeptide of the present disclosure comprises a) i) an epitope, ii) a β2M polypeptide, iii) a first mutant PD-L1 polypeptide of the present disclosure, iv) a second mutant PD of the present disclosure. A L1 polypeptide, and v) a first polypeptide comprising a third variant PD-L1 polypeptide of the present disclosure in N-terminal to C-terminal order; b) i) a class I MHC heavy chain, and ii) A second polypeptide comprising an Fc polypeptide in order from the N-terminus to the C-terminus. In some cases, each of the first mutant PD-L1 polypeptide, the second mutant PD-L1 polypeptide, and the third mutant PD-L1 polypeptide is i) at least relative to the amino acid sequence shown in FIG. 2B. An amino acid sequence having 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 37 is other than threonine For example, amino acid 37 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. For example, amino acid 37 is Gly, Ala, Val, Leu, an amino acid sequence that is le, Arg, Lys, or His, or ii) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, relative to the amino acid sequence set forth in SEQ ID NO: 2. An amino acid sequence having at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 19 is any amino acid other than threonine, eg, amino acid 19 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu, for example, amino acid 19 is Gly, Ala, Val, Leu, Ile, Arg, Lys, or His Roh, including the acid sequence. In some cases, a multimeric polypeptide of the present disclosure comprises a) i) an epitope, and ii) a first polypeptide comprising a β2M polypeptide in N-terminal to C-terminal order, and b) i) the first of the present disclosure. One mutant PD-L1 polypeptide, ii) a second mutant PD-L1 polypeptide of the present disclosure, iii) a third mutant PD-L1 polypeptide of the present disclosure, iv) a class I MHC heavy chain, and v) A second polypeptide comprising an Fc polypeptide in order from the N-terminus to the C-terminus. In some cases, each of the first mutant PD-L1 polypeptide, the second mutant PD-L1 polypeptide, and the third mutant PD-L1 polypeptide is i) at least relative to the amino acid sequence shown in FIG. 2B. An amino acid sequence having 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 37 is other than threonine For example, amino acid 37 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. For example, amino acid 37 is Gly, Ala, Val, Leu, an amino acid sequence that is le, Arg, Lys, or His, or ii) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, relative to the amino acid sequence set forth in SEQ ID NO: 2. An amino acid sequence having at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 19 is any amino acid other than threonine, eg, amino acid 19 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu, for example, amino acid 19 is Gly, Ala, Val, Leu, Ile, Arg, Lys, or His Roh, including the acid sequence. In some cases, a multimeric polypeptide of the present disclosure comprises a) i) an epitope, and ii) a first polypeptide comprising a β2M polypeptide in N-terminal to C-terminal order, and b) i) the first of the present disclosure. 1 mutant PD-L1 polypeptide, ii) linker, iii) second mutant PD-L1 polypeptide of the present disclosure, iv) linker, v) third mutant PD-L1 polypeptide of the present disclosure, vi) class I MHC heavy chain, and vii) a second polypeptide comprising an Fc polypeptide in N-terminal to C-terminal order. In some cases, each of the first mutant PD-L1 polypeptide, the second mutant PD-L1 polypeptide, and the third mutant PD-L1 polypeptide is i) at least relative to the amino acid sequence shown in FIG. 2B. An amino acid sequence having 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 37 is other than threonine For example, amino acid 37 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. For example, amino acid 37 is Gly, Ala, Val, Leu, an amino acid sequence that is le, Arg, Lys, or His, or ii) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, relative to the amino acid sequence set forth in SEQ ID NO: 2. An amino acid sequence having at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 19 is any amino acid other than threonine, eg, amino acid 19 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu, for example, amino acid 19 is Gly, Ala, Val, Leu, Ile, Arg, Lys, or His Roh, including the acid sequence. In some cases, the linker comprises the sequence (GSSSS) _n , where n is 1, 2, 3, 4, or 5. In some cases, n is 4. In some cases, n is 5.

Y56 Substitution In some cases, a multimeric polypeptide of the present disclosure comprises a) i) an epitope, ii) a β2M polypeptide, and iii) at least 70%, at least 75%, at least relative to the amino acid sequence shown in FIG. A mutant PD-L1 polypeptide comprising an amino acid sequence having 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 56 is other than tyrosine For example, amino acid 56 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Or amino acid 56 is Ala, Gly, Val, Leu Or a variant PD-L1 polypeptide in which amino acid 56 is Asp or Glu, or amino acid 56 is Arg, His, or Lys, or at least the amino acid sequence shown in SEQ ID NO: 2 A mutant PD-L1 polypeptide comprising an amino acid sequence having 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. Amino acid 38 is an amino acid other than tyrosine, for example, amino acid 56 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg , His, Asp, or Glu Or a mutated PD-L1 polypeptide in which amino acid 38 is Ala, Gly, Val, Leu, or Ile, amino acid 38 is Asp or Glu, or amino acid 38 is Arg, His, or Lys A first polypeptide comprising N-terminal to C-terminal order; b) i) a class I MHC heavy chain; and ii) a second polypeptide comprising Fc polypeptide in N-terminal to C-terminal order; including. In some cases, a multimeric polypeptide of the present disclosure comprises a) i) an epitope, and ii) a first polypeptide comprising a β2M polypeptide in N-terminal to C-terminal order, and b) i) shown in FIG. 2B. A mutation comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence PD-L1 polypeptide, wherein amino acid 56 is an amino acid other than tyrosine, for example, amino acid 56 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu, or an amino acid A mutant PD-L1 polypeptide, wherein 6 is Ala, Gly, Val, Leu, or Ile, amino acid 56 is Asp or Glu, or amino acid 56 is Arg, His, or Lys, or a sequence Amino acids having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in number 2. A variant PD-L1 polypeptide comprising a sequence, wherein amino acid 38 is an amino acid other than tyrosine; for example, amino acid 38 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Ar g, His, Asp, or Glu; amino acid 38 is Ala, Gly, Val, Leu, or Ile; amino acid 38 is Asp or Glu; or amino acid 38 is Arg, His, Or a mutant PD-L1 polypeptide that is Lys, and ii) a second polypeptide comprising an Fc polypeptide in N-terminal to C-terminal order. In some cases, a multimeric polypeptide of the present disclosure comprises a) i) an epitope, ii) a β2M polypeptide, iii) a first mutant PD-L1 polypeptide of the present disclosure, iv) a second mutant PD of the present disclosure. A L1 polypeptide, and v) a first polypeptide comprising a third variant PD-L1 polypeptide of the present disclosure in N-terminal to C-terminal order; b) i) a class I MHC heavy chain, and ii) A second polypeptide comprising an Fc polypeptide in order from the N-terminus to the C-terminus. In some cases, each of the first mutant PD-L1 polypeptide, the second mutant PD-L1 polypeptide, and the third mutant PD-L1 polypeptide is i) at least relative to the amino acid sequence shown in FIG. 2B. An amino acid sequence having 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 56 is other than tyrosine For example, amino acid 56 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Or amino acid 56 is Ala, Gly, Val, Leu, or I e, amino acid 56 is Asp or Glu, or amino acid 56 comprises an amino acid sequence that is Arg, His, or Lys, or ii) at least relative to the amino acid sequence shown in SEQ ID NO: 2 An amino acid sequence having 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 38 is other than tyrosine For example, the amino acid 38 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Or amino acid 38 is Ala, Gly, Va , Leu, or whether it is Ile, amino acids 38, or is Asp or Glu, or the amino acid 38 comprises Arg, His, or an amino acid sequence that is Lys. In some cases, a multimeric polypeptide of the present disclosure comprises a) i) an epitope, and ii) a first polypeptide comprising a β2M polypeptide in N-terminal to C-terminal order, and b) i) the first of the present disclosure. One mutant PD-L1 polypeptide, ii) a second mutant PD-L1 polypeptide of the present disclosure, iii) a third mutant PD-L1 polypeptide of the present disclosure, iv) a class I MHC heavy chain, and v) A second polypeptide comprising an Fc polypeptide in order from the N-terminus to the C-terminus. In some cases, each of the first mutant PD-L1 polypeptide, the second mutant PD-L1 polypeptide, and the third mutant PD-L1 polypeptide is i) at least relative to the amino acid sequence shown in FIG. 2B. An amino acid sequence having 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 56 is other than tyrosine For example, amino acid 56 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Or amino acid 56 is Ala, Gly, Val, Leu, or I e, amino acid 56 is Asp or Glu, or amino acid 56 comprises an amino acid sequence that is Arg, His, or Lys, or ii) at least relative to the amino acid sequence shown in SEQ ID NO: 2 An amino acid sequence having 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 38 is other than tyrosine For example, the amino acid 38 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Or amino acid 38 is Ala, Gly, Va , Leu, or whether it is Ile, amino acids 38, or is Asp or Glu, or the amino acid 38 comprises Arg, His, or an amino acid sequence that is Lys. In some cases, a multimeric polypeptide of the present disclosure comprises a) i) an epitope, and ii) a first polypeptide comprising a β2M polypeptide in N-terminal to C-terminal order, and b) i) the first of the present disclosure. 1 mutant PD-L1 polypeptide, ii) linker, iii) second mutant PD-L1 polypeptide of the present disclosure, iv) linker, v) third mutant PD-L1 polypeptide of the present disclosure, vi) class I MHC heavy chain, and vii) a second polypeptide comprising an Fc polypeptide in N-terminal to C-terminal order. In some cases, each of the first mutant PD-L1 polypeptide, the second mutant PD-L1 polypeptide, and the third mutant PD-L1 polypeptide is i) at least relative to the amino acid sequence shown in FIG. 2B. An amino acid sequence having 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 56 is other than tyrosine For example, amino acid 56 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Or amino acid 56 is Ala, Gly, Val, Leu, or I e, amino acid 56 is Asp or Glu, or amino acid 56 comprises an amino acid sequence that is Arg, His, or Lys, or ii) at least relative to the amino acid sequence shown in SEQ ID NO: 2 An amino acid sequence having 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 38 is other than tyrosine For example, the amino acid 38 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Or amino acid 38 is Ala, Gly, Va , Leu, or whether it is Ile, amino acids 38, or is Asp or Glu, or the amino acid 38 comprises Arg, His, or an amino acid sequence that is Lys. In some cases, the linker comprises the sequence (GSSSS) _n , where n is 1, 2, 3, 4, or 5. In some cases, n is 4. In some cases, n is 5.

Substitution of G119 In some cases, a multimeric polypeptide of the present disclosure comprises a) i) an epitope, ii) a β2M polypeptide, and iii) at least 70%, at least 75%, at least with respect to the amino acid sequence shown in FIG. A variant PD-L1 polypeptide comprising an amino acid sequence having 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 119 is other than glycine For example, amino acid 119 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Or amino acid 119 is Ala, Val, Leu Or a mutated PD-L1 polypeptide in which amino acid 119 is Arg, His, or Lys, or amino acid 119 is Glu or Asp, or at least the amino acid sequence shown in SEQ ID NO: 2 A mutant PD-L1 polypeptide comprising an amino acid sequence having 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. The amino acid 101 is an amino acid other than glycine. For example, the amino acid 101 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg. , His, Asp, or a mutant PD-L1 poly, which is lu, amino acid 101 is Ala, Val, Leu, or Ile, amino acid 101 is Arg, His, or Lys, or amino acid 101 is Glu or Asp A first polypeptide comprising peptides in N-terminal to C-terminal order; b) i) a class I MHC heavy chain; and ii) a second polypeptide comprising Fc polypeptides in N-terminal to C-terminal order. ,including. In some cases, a multimeric polypeptide of the present disclosure is a) i) an epitope, and ii) a first polypeptide comprising a β2M polypeptide in N-terminal to C-terminal order, b) i) as shown in FIG. 2B. A mutation comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence PD-L1 polypeptide, wherein amino acid 119 is an amino acid other than glycine, for example, amino acid 119 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu, A mutant PD-L1 polypeptide wherein the acid 119 is Ala, Val, Leu, or Ile, the amino acid 119 is Arg, His, or Lys, or the amino acid 119 is Glu or Asp, or a SEQ ID NO: Amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. Wherein the amino acid 101 is an amino acid other than glycine, for example, the amino acid 101 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys. , Met, Asn, Gln, Lys Arg, His, Asp, or Glu, amino acid 101 is Ala, Val, Leu, or Ile, amino acid 101 is Arg, His, or Lys, or amino acid 101 is Glu or A mutant PD-L1 polypeptide that is Asp, and ii) a second polypeptide comprising Fc polypeptides in N-terminal to C-terminal order. In some cases, a multimeric polypeptide of the present disclosure comprises a) i) an epitope, ii) a β2M polypeptide, iii) a first mutant PD-L1 polypeptide of the present disclosure, iv) a second mutant PD of the present disclosure. A L1 polypeptide, and v) a first polypeptide comprising a third variant PD-L1 polypeptide of the present disclosure in N-terminal to C-terminal order; b) i) a class I MHC heavy chain, and ii) A second polypeptide comprising an Fc polypeptide in order from the N-terminus to the C-terminus. In some cases, each of the first mutant PD-L1 polypeptide, the second mutant PD-L1 polypeptide, and the third mutant PD-L1 polypeptide is i) at least relative to the amino acid sequence shown in FIG. 2B. An amino acid sequence having 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 119 is other than glycine For example, amino acid 119 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Or the amino acid 119 is Ala, Val, Leu, or Il Or amino acid 119 is Arg, His, or Lys, or amino acid 119 comprises an amino acid sequence that is Glu or Asp, or ii) at least 70 relative to the amino acid sequence shown in SEQ ID NO: 2 %, At least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 101 is other than glycine For example, amino acid 101 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Or amino acid 101 is Ala, Va , Leu, or whether it is Ile, amino acids 101, Arg, His, or whether it is Lys, or the amino acid 101, containing the amino acid sequence is Glu or Asp. In some cases, a multimeric polypeptide of the present disclosure comprises a) i) an epitope, and ii) a first polypeptide comprising a β2M polypeptide in N-terminal to C-terminal order, and b) i) the first of the present disclosure. One mutant PD-L1 polypeptide, ii) a second mutant PD-L1 polypeptide of the present disclosure, iii) a third mutant PD-L1 polypeptide of the present disclosure, iv) a class I MHC heavy chain, and v) A second polypeptide comprising an Fc polypeptide in order from the N-terminus to the C-terminus. In some cases, each of the first mutant PD-L1 polypeptide, the second mutant PD-L1 polypeptide, and the third mutant PD-L1 polypeptide is i) at least relative to the amino acid sequence shown in FIG. 2B. An amino acid sequence having 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 119 is other than glycine For example, amino acid 119 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Or the amino acid 119 is Ala, Val, Leu, or Il Or amino acid 119 is Arg, His, or Lys, or amino acid 119 comprises an amino acid sequence that is Glu or Asp, or ii) at least 70 relative to the amino acid sequence shown in SEQ ID NO: 2 %, At least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 101 is other than glycine For example, amino acid 101 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Or amino acid 101 is Ala, Va , Leu, or whether it is Ile, amino acids 101, Arg, His, or whether it is Lys, or the amino acid 101, containing the amino acid sequence is Glu or Asp. In some cases, a multimeric polypeptide of the present disclosure comprises a) i) an epitope, and ii) a first polypeptide comprising a β2M polypeptide in N-terminal to C-terminal order, and b) i) the first of the present disclosure. 1 mutant PD-L1 polypeptide, ii) linker, iii) second mutant PD-L1 polypeptide of the present disclosure, iv) linker, v) third mutant PD-L1 polypeptide of the present disclosure, vi) class I MHC heavy chain, and vii) a second polypeptide comprising an Fc polypeptide in N-terminal to C-terminal order. In some cases, each of the first mutant PD-L1 polypeptide, the second mutant PD-L1 polypeptide, and the third mutant PD-L1 polypeptide is i) at least relative to the amino acid sequence shown in FIG. 2B. An amino acid sequence having 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 119 is other than glycine For example, amino acid 119 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Or the amino acid 119 is Ala, Val, Leu, or Il Or amino acid 119 is Arg, His, or Lys, or amino acid 119 comprises an amino acid sequence that is Glu or Asp, or ii) at least 70 relative to the amino acid sequence shown in SEQ ID NO: 2 %, At least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein amino acid 101 is other than glycine For example, amino acid 101 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Or amino acid 101 is Ala, Va , Leu, or whether it is Ile, amino acids 101, Arg, His, or whether it is Lys, or the amino acid 101, containing the amino acid sequence is Glu or Asp. In some cases, the linker comprises the sequence (GSSSS) _n , where n is 1, 2, 3, 4, or 5. In some cases, n is 4. In some cases, n is 5.

  In any of the above embodiments, the mutant PD-L1 polypeptide present in the multimeric polypeptide may comprise a substitution of one of the amino acids shown in FIG. 10 or FIG. Examples include the following. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of D26 of the amino acid sequence shown in FIG. 2B or a substitution of D8 of the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of T37 in the amino acid sequence shown in FIG. 2B or a substitution of T19 in the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of D49 of the amino acid sequence shown in FIG. 2B or a substitution of D31 of the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of L53 of the amino acid sequence shown in FIG. 2B or a substitution of L35 of the amino acid sequence shown in SEQ ID NO: 2. In some cases, the mutant PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure is a substitution of I54 (V54 in mouse PD-L1) of the amino acid sequence shown in FIG. 2B, or the amino acid shown in SEQ ID NO: 2. Contains substitution of I36 in the sequence. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of Y56 in the amino acid sequence shown in FIG. 2B or a substitution of Y38 in the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of Y56 in the amino acid sequence shown in FIG. 2B or a substitution of Y38 in the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of Q66 in the amino acid sequence shown in FIG. 2B or a substitution of Q48 in the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of Q66 in the amino acid sequence shown in FIG. 2B or a substitution of Q48 in the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of E72 of the amino acid sequence shown in FIG. 2B or a substitution of E54 of the amino acid sequence shown in SEQ ID NO: 2. In some cases, the mutant PD-L1 polypeptide present in the multimeric polypeptide of the present disclosure is a substitution of M115 (I115 of mouse PD-L1) of the amino acid sequence shown in FIG. 2B, or the amino acid shown in SEQ ID NO: 2. Includes substitution of M97 in the sequence. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of I116 of the amino acid sequence shown in FIG. 2B, or a substitution of I98 of the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of G119 in the amino acid sequence shown in FIG. 2B or a substitution of G101 in the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of G120 in the amino acid sequence shown in FIG. 2B or a substitution of G102 in the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of G120 in the amino acid sequence shown in FIG. 2B or a substitution of G102 in the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of A121 of the amino acid sequence shown in FIG. 2B or a substitution of A103 of the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of D122 of the amino acid sequence shown in FIG. 2B or a substitution of D104 of the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of Y123 in the amino acid sequence shown in FIG. 2B or a substitution of Y105 in the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of K124 in the amino acid sequence shown in FIG. 2B or a substitution of K106 in the amino acid sequence shown in SEQ ID NO: 2. In some cases, a mutant PD-L1 polypeptide present in a multimeric polypeptide of the present disclosure comprises a substitution of R125 of the amino acid sequence shown in FIG. 2B or a substitution of K107 of the amino acid sequence shown in SEQ ID NO: 2.

Nucleic Acid The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a mutant PD-L1 polypeptide of the present disclosure. The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a PD-L1 fusion polypeptide of the present disclosure.

  The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a multimeric polypeptide of the present disclosure. In some cases, individual polypeptide chains of a multimeric polypeptide of the present disclosure are encoded by separate nucleic acids. In some cases, all polypeptide chains of a multimeric polypeptide of the present disclosure are encoded by a single nucleic acid. In some cases, the first nucleic acid comprises a nucleotide sequence that encodes a first polypeptide of a multimeric polypeptide of the present disclosure, and the second nucleic acid is a second polypeptide of a multimeric polypeptide of the present disclosure. A nucleotide sequence encoding In some cases, a single nucleic acid comprises a nucleotide sequence that encodes a first polypeptide of a multimeric polypeptide of the present disclosure and a second polypeptide of a multimeric polypeptide of the present disclosure.

Separate nucleic acids encoding individual polypeptide chains of a multimeric polypeptide The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a multimeric polypeptide of the present disclosure. As noted above, in some cases, individual polypeptide chains of a multimeric polypeptide of the present disclosure are encoded by separate nucleic acids. In some cases, nucleotide sequences encoding separate polypeptide chains of a multimeric polypeptide of the present disclosure are operably linked to a transcriptional control element, eg, a promoter such as a promoter functional in eukaryotic cells, wherein The promoter may be a constitutive promoter or an inducible promoter. Accordingly, the present disclosure provides a composition comprising a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a nucleotide sequence that encodes a first polypeptide chain of a multimeric polypeptide of the present disclosure. The second nucleic acid comprises a nucleotide sequence that encodes a second polypeptide chain of a multimeric polypeptide of the present disclosure.

  The present disclosure provides a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a nucleotide sequence that encodes a first polypeptide of a multimeric polypeptide of the present disclosure, The polypeptides are in order from N-terminus to C-terminus: a) an epitope (eg, a T cell epitope), b) a first MHC polypeptide, and c) an immunomodulatory polypeptide (eg, a mutant PD-L1 poly of the present disclosure). The second nucleic acid comprises a nucleotide sequence that encodes a second polypeptide of the multimeric polypeptide of the present disclosure, wherein the second polypeptide is a) second in the order of N-terminal to C-terminal. And b) an Ig Fc polypeptide. Suitable T cell epitopes, MHC polypeptides, immunomodulatory polypeptides and Ig Fc polypeptides have already been described. In some cases, the nucleotide sequences encoding the first and second polypeptides are operably linked to transcriptional control elements. In some cases, the transcriptional control element is a promoter that is functional in eukaryotic cells. In some cases, the nucleic acid is present in a separate expression vector.

  The present disclosure provides a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a nucleotide sequence that encodes a first polypeptide of a multimeric polypeptide of the present disclosure, The polypeptide comprises, in order from the N-terminus to the C-terminus, a) an epitope (eg, a T cell epitope), and b) a first MHC polypeptide, wherein the second nucleic acid is the first of the multimeric polypeptides of the present disclosure. A second polypeptide comprising, in order from N-terminal to C-terminal, a) an immunomodulatory polypeptide (eg, a mutant PD-L1 polypeptide of the present disclosure), b) 2 MHC polypeptides, and c) an Ig Fc polypeptide. Suitable T cell epitopes, MHC polypeptides, mutant PD-L1 immunoregulatory polypeptides and Ig Fc polypeptides have already been described. In some cases, the nucleotide sequences encoding the first and second polypeptides are operably linked to transcriptional control elements. In some cases, the transcriptional control element is a promoter that is functional in eukaryotic cells. In some cases, the nucleic acid is present in a separate expression vector.

Nucleic acids encoding two or more polypeptides present in a multimeric polypeptide The present disclosure includes at least a nucleic acid sequence comprising a nucleotide sequence encoding a first polypeptide and a second polypeptide of a multimeric polypeptide of the present disclosure I will provide a. In some cases, when a multimeric polypeptide of the present disclosure includes a first, second, and third polypeptide, the nucleic acid includes a nucleotide sequence that encodes the first, second, and third polypeptide. . In some cases, the nucleotide sequence encoding the first polypeptide and the second polypeptide of the multimeric polypeptide of the present disclosure includes the nucleotide sequence encoding the first polypeptide and the nucleotide encoding the second polypeptide. Contains a proteolytically cleavable linker inserted between the sequences. In some cases, the nucleotide sequence encoding the first polypeptide and the second polypeptide of the multimeric polypeptide of the present disclosure includes the nucleotide sequence encoding the first polypeptide and the nucleotide encoding the second polypeptide. It contains an in-sequence ribosome entry site (IRES) inserted between the sequences. In some cases, the nucleotide sequence encoding the first polypeptide and the second polypeptide of the multimeric polypeptide of the present disclosure includes the nucleotide sequence encoding the first polypeptide and the nucleotide encoding the second polypeptide. Contains a ribosome skipping signal (or cis-acting hydrolase element, CHYSEL) inserted between the sequences. Examples of nucleic acids are described below, wherein a proteolytic cleavable linker is provided between the nucleotide sequence encoding the first polypeptide and the second polypeptide of the multimeric polypeptide of the present disclosure. In any of these embodiments, an IRES or ribosome skipping signal can be used in place of a nucleotide sequence encoding a proteolytically cleavable linker.

  In some cases, the first nucleic acid (eg, recombinant expression vector, mRNA, viral RNA, etc.) comprises a nucleotide sequence that encodes the first polypeptide chain of a multimeric polypeptide of the present disclosure, and the second nucleic acid (Eg, a recombinant expression vector, mRNA, viral RNA, etc.) comprises a nucleotide sequence that encodes a second polypeptide chain of a multimeric polypeptide of the present disclosure. In some cases, the nucleotide sequence encoding the first polypeptide and the second nucleotide sequence encoding the second polypeptide are each a transcriptional control element, eg, a promoter such as a promoter functional in eukaryotic cells. Operatively linked, where the promoter may be a constitutive promoter or an inducible promoter.

  The present disclosure provides a nucleic acid comprising a nucleotide sequence that encodes a recombinant polypeptide, wherein the recombinant polypeptide comprises, in order from N-terminal to C-terminal, a) an epitope (eg, a T cell epitope), b) A first MHC polypeptide, c) an immunomodulatory polypeptide (eg, a mutant PD-L1 polypeptide of the present disclosure), d) a proteolytic cleavable linker, e) a second MHC polypeptide, and f) immunity Globulin (Ig) Fc polypeptides. The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a recombinant polypeptide, wherein the recombinant polypeptide comprises a) a first leader peptide, b) an epitope, c, in order from N-terminus to C-terminus. A) a first MHC polypeptide, d) an immunomodulatory polypeptide (eg, a mutant PD-L1 polypeptide of the present disclosure), e) a proteolytically cleavable linker, f) a second leader peptide, g) a second And H) an Ig Fc polypeptide. The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a recombinant polypeptide, wherein the recombinant polypeptide comprises, in order from N-terminal to C-terminal, a) an epitope, b) a first MHC polypeptide, c) a proteolytic cleavable linker, d) an immunomodulatory polypeptide (eg, a mutant PD-L1 polypeptide of the present disclosure), e) a second MHC polypeptide, and f) an Ig Fc polypeptide. In some cases, the first leader peptide and the second leader peptide are β2-M leader peptides. In some cases, the nucleotide sequence is operably linked to a transcriptional control element. In some cases, the transcriptional control element is a promoter that is functional in eukaryotic cells.

  Suitable MHC polypeptides have already been described. In some cases, the first MHC polypeptide is a β2-microglobulin polypeptide and the second MHC polypeptide is an MHC class I heavy chain polypeptide. In some cases, the β2-microglobulin polypeptide is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% relative to any one of the amino acid sequences shown in FIG. Amino acid sequences having the same amino acid sequence identity. In some cases, the MHC class I heavy chain polypeptide is an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K or HLA-L heavy chain. In some cases, the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence shown in one of FIGS. In some cases, the first MHC polypeptide is an MHC class II alpha chain polypeptide and the second MHC polypeptide is an MHC class II beta chain polypeptide.

  Suitable Fc polypeptides have already been described. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide, IgG2 Fc polypeptide, IgG3 Fc polypeptide, IgG4 Fc polypeptide, IgA Fc polypeptide or IgM Fc polypeptide. In some cases, the Ig Fc polypeptide has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIGS. An amino acid sequence having

  Suitable mutant PD-L1 immunomodulatory polypeptides have already been described.

  Suitable linkers that can be cleaved by proteolysis have already been described. In some cases, the proteolytic cleavable linker is a) LEVLFQGP (SEQ ID NO: 34), b) ENLYTQS (SEQ ID NO: 35), c) DDDDK (SEQ ID NO: 36), d) LVPR (SEQ ID NO: 37), and e) comprising an amino acid sequence selected from GSGATNFSLLKQAGDVEENGPP (SEQ ID NO: 38).

  In some cases, the linker between the epitope and the first MHC polypeptide includes a first Cys residue, and the second MHC polypeptide includes an amino acid substitution to provide a second Cys residue; As a result, the first and second Cys residues provide a disulfide bond between the linker and the second MHC polypeptide. In some cases, the first MHC polypeptide includes an amino acid substitution to provide a first Cys residue, and the second MHC polypeptide includes an amino acid substitution to provide a second Cys residue, resulting in The first Cys residue and the second Cys residue provide a disulfide bond between the first MHC polypeptide and the second MHC polypeptide.

Recombinant Expression Vector The present disclosure provides a recombinant expression vector comprising a nucleic acid of the present disclosure. In some cases, the recombinant expression vector is a non-viral vector. In some embodiments, the recombinant expression vector is a viral construct, such as a recombinant adeno-associated virus construct (see, eg, US Pat. No. 7,078,387), a recombinant adenoviral construct, a recombinant lenti. Such as viral constructs, recombinant retroviral constructs, non-integrating viral vectors.

  Suitable expression vectors include, but are not limited to, viral vectors (eg, vaccinia virus, poliovirus, adenovirus (eg, Li et al., Invest Optalmol Vi Sci 35: 2543 2549, 1994, Borras et al., Gene Ther). 6: 515 524, 1999, Li and Davidson, PNAS 92: 7700 7704, 1995, Sakamoto et al., H Gene Ther 5: 1088 1097, 1999, WO 94/12649, WO 93/03769, WO 93/19191, WO 94/28938, WO 95/11984 and WO 95/00655), adeno-associated viruses (see, eg, Ali et al., um Gene Ther 9: 886, 1998, Flannery et al., PNAS 94: 6916 6921, 1997, Bennett et al., Invest Optalmol Vis Sci 38: 2857 2863, 1997, Journal et al. 6: 90, J. et al. , 1997, Rolling et al., Hum Gene Ther 10: 641 648, 1999, Ali et al., Hum Mol Genet 5: 591 594, 1996, Srivastava in WO 93/09239, Samulski et al. ) 63: 3822 3828, Mendelson et al., Virol. (1988) 166: 154-165, and Flotte et al., PNAS (1993) 90: 10613 10617), SV40, herpes simplex virus, human immunodeficiency virus (eg, Miyoshi et al., PNAS 94: 10319 23, 1997, Viral vectors based on Takahashi et al., J Virol 73: 7812 7816, 1999), retroviral vectors (eg, murine leukemia virus, spleen necrosis virus, and Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus). , Vectors derived from retroviruses such as lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and breast tumor virus).

  Many suitable expression vectors are known to those skilled in the art and many are commercially available. By way of example, the following vectors are provided: For eukaryotic host cells: pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG and pSVLSV40 (Pharmacia). However, any other vector can be used as long as it is compatible with the host cell.

  Depending on the host / vector system utilized, several suitable transcriptional and translational control elements can be used in the expression vector, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. (eg, Bitter et al. (1987) Methods in Enzymology, 153: 516-544).

  In some embodiments, the nucleotide sequence encoding the DNA targeted RNA and / or site specific modified polypeptide is operably linked to a regulatory element, eg, a transcriptional regulatory element such as a promoter. Transcriptional control elements can be functional in either eukaryotic cells, eg, mammalian cells, or prokaryotic cells (eg, bacterial cells or archaeal cells). In some embodiments, the nucleotide sequence encoding the DNA-targeting RNA and / or site-specific modified polypeptide is prokaryotic for expression of the nucleotide sequence encoding the DNA-targeted RNA and / or site-specific modified polypeptide. And operably linked to a plurality of control elements that enable both eukaryotic cells.

  Non-limiting examples of suitable eukaryotic promoters (promoter functional in eukaryotic cells) include cytomegalovirus (CMV) early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, retroviral long Examples include terminal repeat (LTR) and those derived from mouse metallothionein-I. Selection of appropriate vectors and promoters is well within the level of ordinary skill in the art. The expression vector can also include a ribosome binding site for translation initiation and a transcription terminator. Expression vectors can also include appropriate sequences for amplifying expression.

Genetically modified host cell The present disclosure provides a genetically modified host cell wherein the host cell is genetically modified with a nucleic acid of the present disclosure.

  Suitable host cells include eukaryotic cells such as yeast cells, insect cells and mammalian cells. In some cases, the host cell is a cell of a mammalian cell line. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (eg, mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (eg, American Cultured Cell Line Conservation Organization (ATCC) number CCL-2), Chinese hamster ovary (CHO) cells (eg, ATCC numbers CRL9618, CCL61, CRL9096). 293 cells (eg, ATCC number CRL-1573), Vero cells, NIH3T3 cells (eg, ATCC number CRL-1658), Huh-7 cells, BHK cells (eg, ATCC number CCL10), PC12 cells (ATCC number CRL1721) , COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human fetal kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, etc.

  In some cases, the host cell is a mammalian cell that has been genetically modified to not synthesize endogenous MHCβ2-M.

Methods for Producing Multimeric Polypeptides The present disclosure provides methods for producing multimeric polypeptides of the present disclosure. This method generally involves culturing a host cell that has been genetically modified with a recombinant expression vector comprising a nucleotide sequence encoding a multimeric polypeptide in a culture medium and a multimer from the genetically modified host cell and / or culture medium. Isolating the polypeptide. A host cell that has been genetically modified with a recombinant expression vector comprising a nucleotide sequence encoding a multimeric polypeptide is also referred to as an “expression host”. As noted above, in some cases, individual polypeptide chains of a multimeric polypeptide of the present disclosure are encoded in separate recombinant expression vectors. In some cases, all polypeptide chains of a multimeric polypeptide of the present disclosure are encoded in a single recombinant expression vector.

  Isolation of multimeric polypeptides from expression host cells (eg, lysates of expression host cells) and / or culture medium in which the host cells are cultured can be performed using standard methods of protein purification.

  For example, a lysate can be prepared from an expression host and the lysate can be purified using high performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography or other purification techniques. Alternatively, if the multimeric polypeptide is secreted from the expression host cell into the culture medium, the multimeric polypeptide is cultured using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. It can be purified from the medium. In some cases, the composition used is at least 80% by weight, at least about 85% by weight, at least about 95% by weight or at least about 99% by weight compared to contaminants related to the method of preparation of the product and its purification. Contains 5% by weight of the desired product. The percentage can be based on total protein.

  In some cases, for example, if the multimeric polypeptide includes an affinity tag, the binding tag to which the affinity tag is immobilized can be used to purify the multimeric polypeptide.

Compositions The present disclosure provides compositions comprising a mutant PD-L1 polypeptide of the present disclosure, including pharmaceutical compositions. The present disclosure provides compositions comprising a multimeric polypeptide of the present disclosure, including pharmaceutical compositions. The present disclosure provides compositions comprising the nucleic acids or recombinant expression vectors of the present disclosure, including pharmaceutical compositions.

Compositions Comprising Multimeric Polypeptides In addition to the multimeric polypeptides of the present disclosure, the compositions of the present disclosure can include one or more of the following: salts, eg, NaCl, MgCl ₂ , KCl, MgSO ₄ , buffering agents such as Tris buffer, N- (2-hydroxyethyl) piperazine-N ′-(2-ethanesulfonic acid) (HEPES), 2- (N-morpholino) ethanesulfonic acid (MES), 2- (N-morpholino) ethanesulfonic acid sodium salt (MES), 3- (N-morpholino) propanesulfonic acid (MOPS), N-tris [hydroxymethyl] methyl-3-aminopropanesulfonic acid (TAPS), etc. Solubilizers, detergents such as non-ionic detergents such as Tween-20, protease inhibitors, glycerol Such.

  The composition can include pharmaceutically acceptable excipients, and various pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients are described, for example, in “Remington: The Science and Practice of Pharmacy”, 19th Ed. (1995) or the latest edition, Mack Publishing Co, A.M. Gennaro (2000) “Remington: The Science and Practice of Pharmacy”, 20th edition, Lipcott, Williams, & Wilkins, Pharmaceutical Dosage FormsDrug. C. Ansel et al. Eds 7th ed. , Lippincott, Williams, & Wilkins, and Handbook of Pharmaceutical Excipients (2000) A.A. H. Kibbe et al. , Eds. , 3rd ed. Amer. Fully described in various publications, including Pharmaceutical Assoc.

  The pharmaceutical composition can comprise a multimeric polypeptide of the present disclosure and a pharmaceutically acceptable excipient. In some cases, a subject pharmaceutical composition is suitable for administration to a subject, eg, sterile. For example, in some embodiments, a subject pharmaceutical composition is suitable for administration to a human subject, for example, where the composition is sterile and includes detectable pyrogens and / or other toxins. Not.

  The protein composition can include other ingredients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcan, cellulose, glucose, sucrose, magnesium, carbonate, and the like. The composition comprises pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents such as sodium acetate, sodium chloride, potassium chloride, chloride. Calcium, sodium lactate, hydrochloride, sulfate, solvates (eg, mixed ionic salts, water, organics), hydrates (eg, water), and the like can be included.

  For example, the composition can include aqueous solutions, powders, granules, tablets, pills, suppositories, capsules, suspensions, sprays, and the like. The composition can be formulated according to various administration routes described below.

  When the multimeric polypeptide of the present disclosure is administered as an injection directly into tissue (eg, subcutaneously, intraperitoneally, intramuscularly and / or intravenously), the formulation is a ready-to-use dosage form Or as a non-aqueous form (eg, a reconstitutable storage stable powder) or an aqueous form, eg, a solution composed of pharmaceutically acceptable carriers and excipients. Protein-containing formulations can also be provided to increase the serum half-life of the protein of interest after administration. For example, the protein can be provided in a liposomal formulation prepared as a colloid, or other conventional technique for extending serum half-life. For example, Szoka et al. 1980 Ann. Rev. Biophys. Bioeng. 9: 467, U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028, various methods are available for preparing liposomes. is there. The preparation can also be provided in a controlled release or sustained release form.

  Other examples of formulations suitable for parenteral administration include isotonic sterile injections, antioxidants, bacteriostats and solutes, suspending agents, solubilizing agents that make the formulation isotonic with the blood of the intended recipient. Agents, thickeners, stabilizers and preservatives. For example, the subject pharmaceutical composition may be present in a container, such as a sterile container, such as a syringe. The formulation can be provided in unit dose or multi-dose closed containers such as ampoules and vials, which are freeze-dried that require only sterile liquid excipients such as water to be added immediately prior to use for injection. It can be stored in (freeze-dried) state. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

  The concentration of the multimeric polypeptide of the present disclosure in the formulation varies widely (eg, less than about 0.1% (usually about 2% or at least 2%) to about 20% to 50% or on a weight basis, or And more) and is usually selected based primarily on fluid volume, viscosity, and patient-based factors, depending on the particular mode of administration selected and patient requirements.

  The present disclosure provides a container comprising a composition of the present disclosure, eg, a liquid composition. The container may be, for example, a syringe or an ampoule. In some cases, the container is sterile. In some cases, both the container and the composition are sterile.

  The present disclosure provides compositions comprising a mutant PD-L1 polypeptide of the present disclosure, including pharmaceutical compositions. The composition can comprise the excipients described above for a) a mutant PD-L1 polypeptide of the present disclosure, and b) a multimeric polypeptide. In some cases, the excipient is a pharmaceutically acceptable excipient.

Compositions Comprising Nucleic Acids or Recombinant Expression Vectors The present disclosure provides compositions, eg, pharmaceutical compositions, comprising nucleic acids or recombinant expression vectors of the present disclosure. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients are, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy”, 20th edition, Lipcott, Williams, & Wilkins, Pharmaceutical Dosage FormsDrug. C. Ansel et al. Eds 7th ed. , Lippincott, Williams, & Wilkins, and Handbook of Pharmaceutical Excipients (2000) A.A. H. Kibbe et al. , Eds. , 3rd ed. Amer. It is well documented in various publications, including Pharmaceutical Assoc.

The composition of the present disclosure comprises a) a nucleic acid or recombinant expression vector of interest, and b) a buffer, a surfactant, an antioxidant, a hydrophilic polymer, a dextrin, a chelating agent, a suspending agent, a solubilizing agent, One or more of thickeners, stabilizers, bacteriostats, wetting agents and preservatives can be included. Suitable buffers include, but are not limited to, (for example, N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), bis (2-hydroxyethyl) amino-tris (hydroxymethyl) Methane (bis-tris), N- (2-hydroxyethyl) piperazine-N′3-propanesulfonic acid (EPPS or HEPPS), glycylglycine, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), 3- (N-morpholino) propanesulfonic acid (MOPS), piperazine-N, N′-bis (2-ethane-sulfonic acid) (PIPES), sodium bicarbonate, 3- (N-tris (hydroxy) Methyl) -methyl-amino) -2-hydroxy-propanesulfonic acid) TAPSO, (N-tris (hydroxy) ) Methyl-2-aminoethanesulfonic acid (TES), N-tris (hydroxymethyl) methyl - glycine (Tricine), tris (hydroxymethyl) -, etc. aminomethane (Tris)) can be mentioned. Suitable salts include, for example, NaCl, MgCl ₂ , KCl, MgSO _{4 and the} like.

  The pharmaceutical formulations of the present disclosure can comprise a nucleic acid or recombinant expression vector of the present disclosure in an amount of about 0.001% to about 90% (w / w). In the description of the formulations below, “subject nucleic acid or recombinant expression vector” is understood to include a nucleic acid or recombinant expression vector of the present disclosure. For example, in some embodiments, a subject formulation comprises a nucleic acid or recombinant expression vector of the present disclosure.

  The subject nucleic acid or recombinant expression vector can be mixed, encapsulated, conjugated or conjugated with other compounds or mixtures of compounds, such as liposomes or receptor targeting molecules Can be mentioned. The subject nucleic acids or recombinant expression vectors can be combined in the formulation with one or more components that aid in uptake, distribution and / or absorption.

  The composition of the subject nucleic acid or recombinant expression vector includes, but is not limited to, many of the possible dosage forms such as tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories and enemas. Either can be formulated. The subject nucleic acid or recombinant expression vector compositions can also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further comprise substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain a stabilizer.

  The preparation containing the subject nucleic acid or recombinant expression vector may be a liposome preparation. As used herein, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer (s). Liposomes are unilamellar or multilamellar vesicles with an aqueous interior that includes a membrane formed from a lipophilic material and the composition to be delivered. Cationic liposomes are positively charged liposomes that can interact with negatively charged DNA molecules to form stable complexes. Liposomes that are pH sensitive or negatively charged are believed to encapsulate DNA rather than complex with DNA. Both cationic and non-cationic liposomes can be used to deliver the subject nucleic acids or recombinant expression vectors.

  Liposomes also include “sterically stabilized” liposomes, and as used herein, the term enhances circulation lifetime when incorporated into liposomes as compared to liposomes that lack specialized lipids. Refers to a liposome containing a species or multiple specialized lipids. Examples of sterically stabilized liposomes include one or more glycolipids in which a portion of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids, or one or more hydrophilic properties such as a polyethylene glycol (PEG) portion. It has been derivatized with a polymer. Liposomes and their uses are further described in US Pat. No. 6,287,860, which is hereby incorporated by reference in its entirety.

  The formulations and compositions of the present disclosure can also include a surfactant. The use of surfactants in drug products, formulations and emulsions is well known in the art. Surfactants and their use are further described in US Pat. No. 6,287,860.

  In one embodiment, various penetration enhancers are included to provide efficient delivery of nucleic acids. In addition to helping the non-lipophilic drug diffuse across the cell membrane, penetration enhancers also increase the permeability of the lipophilic drug. Penetration enhancers can be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents and non-chelating non-surfactants. Penetration enhancers and their use are further described in US Pat. No. 6,287,860, which is incorporated herein by reference in its entirety.

  Compositions and preparations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets Or a mini-tablet is mentioned. Thickeners, flavoring agents, diluents, emulsifiers, dispersion aids or binders may be desirable. Suitable oral formulations include those in which the subject antisense nucleic acids are administered with one or more penetration enhancers, surfactants and chelating agents. Suitable surfactants include, but are not limited to, fatty acids and / or esters or salts thereof, bile acids and / or salts thereof. Suitable bile acids / salts and fatty acids and their use are further described in US Pat. No. 6,287,860. Combinations of penetration enhancers are also suitable, for example fatty acids / salts in combination with bile acids / salts. An exemplary suitable combination is the sodium salt of lauric acid, capric acid and UDCA. Additional penetration enhancers include, but are not limited to, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. Suitable penetration enhancers also include propylene glycol, dimethyl sulfoxide, triethanolamine, N, N-dimethylacetamide, N, N-dimethylformamide, 2-pyrrolidone and their derivatives, tetrahydrofurfuryl alcohol and AZONE ™. Can be mentioned.

Therapeutic Methods The present invention provides a method for selectively modulating the activity of epitope-specific T cells in an individual, wherein the method comprises an amount effective to selectively modulate the activity of epitope-specific T cells in the individual. Administering to the individual a multimeric polypeptide of the present disclosure or one or more nucleic acids encoding the multimeric polypeptide. In some cases, the therapeutic methods of the present disclosure include administering one or more recombinant expression vectors comprising a nucleotide sequence encoding a multimeric polypeptide of the present disclosure to an individual in need thereof. In some cases, the therapeutic methods of the present disclosure include administering one or more mRNA molecules comprising a nucleotide sequence encoding a multimeric polypeptide of the present disclosure to an individual in need thereof. In some cases, the therapeutic methods of the present disclosure include administering the multimeric polypeptides of the present disclosure to an individual in need thereof.

  The present disclosure provides a method of selectively modulating the activity of epitope-specific T cells in an individual, wherein the method comprises an effective amount of a multimeric polypeptide of the present disclosure or a nucleotide sequence encoding a multimeric polypeptide. Administering to the individual one or more nucleic acids (eg, expression vectors, mRNA, etc.), wherein the multimeric polypeptide selectively modulates the activity of epitope-specific T cells in the individual. Selectively modulating the activity of epitope-specific T cells can treat a disease or disorder in an individual. Accordingly, the present disclosure provides a method of treatment comprising administering an effective amount of a multimeric polypeptide of the present disclosure to an individual in need thereof.

  In some cases, an immunomodulatory polypeptide (eg, a mutant PD-L1 polypeptide of the present disclosure) present in a multimeric polypeptide of the present disclosure is a suppressor polypeptide and a multimeric polypeptide comprising a mutant PD-L1 polypeptide Suppresses the activity of epitope-specific T cells. In some cases, the epitope is a self-epitope and the multimeric polypeptide selectively inhibits the activity of T cells specific for the self-epitope.

  The present disclosure provides a method of treating an autoimmune disorder in an individual, the method comprising an effective amount of one or more nucleic acids comprising a multimeric polypeptide of the present disclosure or a nucleotide sequence encoding a multimeric polypeptide. Wherein the multimeric polypeptide includes a T cell epitope that is a self-epitope, and the multimeric polypeptide includes a mutant PD-L1 polypeptide of the present disclosure. In some cases, an “effective amount” of a multimeric polypeptide is administered before or with the administration of the multimeric polypeptide when administered in one or more doses to an individual in need thereof. At least 10%, at least 15%, at least 20%, at least 25% of the number and / or activity of autoreactive T cells compared to the number and / or activity of autoreactive T cells in an individual free of An amount that reduces by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%. In some cases, an “effective amount” of multimeric polypeptides is an amount that reduces the production of Th2 cytokines in an individual when administered in one or more doses to an individual in need thereof. In some cases, an “effective amount” of multimeric polypeptides ameliorates one or more symptoms associated with an autoimmune disease in an individual when administered in one or more doses to the individual in need thereof. Amount.

  Suitable autoimmune disorders for treatment with the methods of the present disclosure include, but are not limited to, alopecia areata, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune Addison disease, adrenal autoimmune disease, autoimmunity Hemolytic anemia, autoimmune hepatitis, autoimmune ovitis and testitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immunodeficiency syndrome ( CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, scarring pemphigus, CREST syndrome (also known as limited cutaneous systemic sclerosis), cold agglutinin disease, Crohn's disease, Discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain Valley, Hashimoto's thyroiditis, idiopathic pulmonary line , Idiopathic thrombocytopenic purpura (ITP), irritable bowel disease (IBD), IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, meniere disease, mixed connective tissue disease, multiple Sclerosis, type 1 diabetes, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, Primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff man syndrome, systemic lupus erythematosus, lupus erythematosus, Takayasu Arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis (herpes Dermatitis vasculitis (dermatitis herpetiformis vasculitis), etc.), vitiligo, and Wegener's granulomatosis, and the like.

  In some cases, an immunomodulatory polypeptide (eg, a mutant PD-L1 polypeptide of the present disclosure) present in a multimeric polypeptide of the present disclosure is a suppressor polypeptide and a multimeric polypeptide comprising a mutant PD-L1 polypeptide Suppresses the activity of epitope-specific T cells. In some cases, the epitope is an allograft (eg, skin allograft, liver allograft, kidney allograft, heart allograft, bone allograft, cartilage allograft, lung allograft, cell allograft An epitope present in a graft (eg, bone marrow allograft), and the multimeric polypeptide selectively suppresses the activity of T cells specific for the antigen present in the allograft.

  The present disclosure provides a method of inhibiting allograft rejection in an individual, wherein the method is for an individual (e.g., an individual who is an allograft recipient, or an individual who will soon be an allograft recipient), Administering an effective amount of a multimeric polypeptide of the present disclosure, or one or more nucleic acids comprising a nucleotide sequence encoding the multimeric polypeptide, wherein the multimeric polypeptide is an epitope present in an allograft In addition to containing certain T cell epitopes, multimeric polypeptides include mutant PD-L1 polypeptides of the present disclosure. In some cases, an “effective amount” of a multimeric polypeptide, when administered to an individual in need thereof at one or more doses, the individual prior to administration of the multimeric polypeptide, or the multimeric polypeptide The number and / or activity of alloreactive (allograft reactive) T cells is at least 10 compared to the number and / or activity of alloreactive (allograft reactive) T cells in an individual who is not administered. %, At least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% is there. In some cases, an “effective amount” of a multimeric polypeptide is an amount that, when administered at one or more doses to an individual in need thereof, increases the survival time of the allograft in the individual, eg, The survival time of the allograft in the individual is at least 25%, at least 50%, at least 2 times, at least 5 times, at least 10 times, compared to the survival time of the allograft in the individual to which the multimeric polypeptide is not administered. Extend at least 50 times, or at least 100 times. In some cases, an “effective amount” of a multimeric polypeptide, when administered at one or more doses to an individual in need thereof, exhibits one or more symptoms associated with allograft rejection in the individual. The amount to remit.

  As noted above, in some cases, in the practice of a subject treatment method, the multimeric polypeptides of the present disclosure are administered to an individual in need thereof as a polypeptide. In other cases, in the practice of a subject treatment method, nucleic acids comprising one or more nucleotide sequences encoding a multimeric polypeptide of the present disclosure are administered to an individual in need thereof. Accordingly, in other cases, one or more nucleic acids of the present disclosure, eg, one or more recombinant expression vectors of the present disclosure, is administered to an individual in need thereof.

Formulations Suitable formulations have already been described, and suitable formulations contain pharmaceutically acceptable excipients. In some cases, a suitable formulation comprises a) a multimeric polypeptide of the present disclosure, and b) a pharmaceutically acceptable excipient. In some cases, a suitable formulation comprises a) a nucleic acid comprising a nucleotide sequence encoding a multimeric polypeptide of the present disclosure, and b) a pharmaceutically acceptable excipient, in some cases the nucleic acid is mRNA. It is. In some cases, suitable formulations include: a) a first nucleic acid comprising a nucleotide sequence that encodes a first polypeptide of a multimeric polypeptide of the present disclosure; b) a second poly of a multimeric polypeptide of the present disclosure. A second nucleic acid comprising a nucleotide sequence encoding the peptide, and c) a pharmaceutically acceptable excipient. In some cases, a suitable formulation comprises a) a recombinant expression vector comprising a nucleotide sequence encoding a multimeric polypeptide of the present disclosure, and b) a pharmaceutically acceptable excipient. In some cases, suitable formulations include: a) a first recombinant expression vector comprising a nucleotide sequence encoding the first polypeptide of the disclosed multimeric polypeptide, b) the first of the multimeric polypeptide of the present disclosure. A second recombinant expression vector comprising a nucleotide sequence encoding two polypeptides, and c) a pharmaceutically acceptable excipient.

  Suitable excipients that are pharmaceutically acceptable have already been described.

Dosage The appropriate dosage can be determined by the attending physician or other qualified medical personnel based on various clinical factors. As is well known in the medical field, the dosage for any given patient will depend on the patient's size, body surface area, age, the specific polypeptide or nucleic acid administered, patient sex, time and route of administration, overall Depends on many factors, including health status and other drugs administered at the same time. A multimeric polypeptide of the present disclosure can have between 1 ng / kg body weight and 20 mg / kg body weight per administration, such as between 0.1 mg / kg body weight and 10 mg / kg body weight, such as between 0.5 mg / kg body weight and 5 mg / kg body weight. However, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. If the regimen is continuous infusion, this may also be in the range of 1 μg to 10 mg / kg body weight / minute.

  In some cases, suitable doses of multimeric polypeptides of the present disclosure are 0.01 μg to 100 g / kg body weight, 0.1 μg to 10 g / kg body weight, 1 μg to 1 g / kg body weight, 10 μg to 100 mg / kg body weight, 100 μg. 10 mg / kg body weight or 100 μg to 1 mg / kg body weight. Based on the measured residence time and concentration of the administered drug in the body fluid or tissue, one skilled in the art can easily estimate the repetition rate of the dosing. Following successful treatment, it may be desirable to have the patient receive maintenance therapy to prevent recurrence of the condition, wherein the multimeric polypeptide of the present disclosure is between 0.01 μg and 100 g / kg body weight. , 0.1 μg to 10 g / kg body weight, 1 μg to 1 g / kg body weight, 10 μg to 100 mg / kg body weight, 100 μg to 10 mg / kg body weight, or 100 μg to 1 mg / kg body weight.

  One skilled in the art will readily recognize that dose levels can vary depending on the particular multimeric polypeptide, the severity of the symptoms, and the subject's susceptibility to side effects. Preferred dosages for a given compound are readily determined by those skilled in the art by various means.

  In some embodiments, multiple doses of a multimeric polypeptide of the present disclosure, a nucleic acid of the present disclosure or a recombinant expression vector of the present disclosure are administered. The frequency of administration of the multimeric polypeptide of the present disclosure, the nucleic acid of the present disclosure or the recombinant expression vector of the present disclosure may vary depending on any of a variety of factors, such as the severity of symptoms. For example, in some embodiments, a multimeric polypeptide of the present disclosure, a nucleic acid of the present disclosure or a recombinant expression vector of the present disclosure may be once a month, twice a month, three times a month, every other week (qow), week 1 Times (qw), twice a week (biw), three times a week (tiw), four times a week, five times a week, six times a week, every other day (qod), once a day (qd), twice a day (qid) ) Or three times daily (tid).

  Duration of administration of a multimeric polypeptide of the present disclosure, a nucleic acid of the present disclosure or a recombinant expression vector of the present disclosure, eg, administration of a multimeric polypeptide of the present disclosure, a nucleic acid of the present disclosure or a recombinant expression vector of the present disclosure. The time period taken may vary depending on any of a variety of factors, such as patient response. For example, a multimeric polypeptide of the present disclosure, a nucleic acid of the present disclosure or a recombinant expression vector of the present disclosure can be about 1 day to about 1 week, about 2 weeks to about 4 weeks, about 1 month to about 2 months, About 2 months to about 4 months, about 4 months to about 6 months, about 6 months to about 8 months, about 8 months to about 1 year, about 1 year to about 2 years, or about 2 It can be administered over a period ranging from years to about 4 years or more.

Route of Administration The active agent (multimeric polypeptide of the present disclosure, nucleic acid of the present disclosure or recombinant expression vector of the present disclosure) may be any available method and route suitable for drug delivery, including in vivo and ex vivo methods. And systemic and local routes of administration.

  Conventional pharmaceutically acceptable routes of administration include intratumoral, peritumoral, intramuscular, intratracheal, intracranial, subcutaneous, intradermal, topical application, intravenous, intraarterial, rectal, nasal, oral and others Enteral and parenteral routes of administration. The routes of administration can be combined or adjusted as needed depending on the multimeric polypeptide and / or the desired effect. A multimeric polypeptide of the present disclosure or a nucleic acid or recombinant expression vector of the present disclosure can be administered in a single dose or multiple doses.

  In some embodiments, a multimeric polypeptide of the present disclosure, a nucleic acid of the present disclosure or a recombinant expression vector of the present disclosure is administered intravenously. In some embodiments, a multimeric polypeptide of the present disclosure, a nucleic acid of the present disclosure or a recombinant expression vector of the present disclosure is administered intramuscularly. In some embodiments, a multimeric polypeptide of the present disclosure, a nucleic acid of the present disclosure or a recombinant expression vector of the present disclosure is administered locally. In some embodiments, a multimeric polypeptide of the present disclosure, a nucleic acid of the present disclosure or a recombinant expression vector of the present disclosure is administered intratumorally. In some embodiments, a multimeric polypeptide of the present disclosure, a nucleic acid of the present disclosure or a recombinant expression vector of the present disclosure is administered around a tumor. In some embodiments, a multimeric polypeptide of the present disclosure, a nucleic acid of the present disclosure or a recombinant expression vector of the present disclosure is administered intracranially. In some embodiments, a multimeric polypeptide of the present disclosure, a nucleic acid of the present disclosure or a recombinant expression vector of the present disclosure is administered subcutaneously.

  In some embodiments, multimeric polypeptides of this disclosure are administered intravenously. In some embodiments, multimeric polypeptides of the present disclosure are administered intramuscularly. In some embodiments, multimeric polypeptides of the present disclosure are administered locally. In some embodiments, multimeric polypeptides of the present disclosure are administered intratumorally. In some embodiments, multimeric polypeptides of the present disclosure are administered around the tumor. In some embodiments, multimeric polypeptides of the present disclosure are administered intracranially. In some embodiments, the multimeric polypeptide is administered subcutaneously.

  The multimeric polypeptide of the present disclosure, the nucleic acid of the present disclosure or the recombinant expression vector of the present disclosure may be any available conventional method and route suitable for delivery of conventional drugs, including systemic or local routes. Can be used to administer to a host. In general, administration routes contemplated by the present invention include, but are not necessarily limited to, enteral, parenteral or inhalation routes.

  Parenteral routes of administration other than inhalation are not necessarily limited, but are topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intrathecal, intrasternal, intratumoral, peritumoral and intravenous Routes, ie any route of administration other than via the gastrointestinal tract. Parenteral administration can be performed to provide systemic or local delivery of the multimeric polypeptide of the present disclosure, the nucleic acid of the present disclosure or the recombinant expression vector of the present disclosure. Where systemic delivery is desired, administration typically involves topical or mucosal administration of a pharmaceutical preparation that is absorbed invasively or systemically.

Subjects suitable for treatment Subjects suitable for treatment by the methods of the present disclosure include individuals diagnosed with cancer, individuals who have been treated for cancer but have failed to respond to treatment, and those who have been treated for cancer. , Individuals with cancer, including those who responded early but subsequently became resistant to treatment. Subjects suitable for treatment by the methods of the present disclosure include infections (eg, pathogens such as, for example, individuals diagnosed as having an infection, and individuals who have been treated for infection but have failed to respond to treatment). Individuals having infection with bacteria, viruses, protozoa, etc.). Subjects suitable for treatment by the methods of the present disclosure include individuals with a bacterial infection, including individuals diagnosed with a bacterial infection and individuals who have been treated with a bacterial infection but have failed to respond to the treatment. Can be mentioned. Subjects suitable for treatment by the methods of the present disclosure include individuals with a viral infection, including individuals diagnosed with a viral infection and individuals who have been treated with a viral infection but have failed to respond to the treatment. Can be mentioned. Subjects suitable for treatment by the methods of the present disclosure include autoimmune diseases, including individuals diagnosed as having autoimmune disease and individuals who have been treated but have failed to respond to treatment. An individual having it is mentioned.

  The following examples are provided to fully disclose and explain the methods of making and using the present invention to those skilled in the art and are intended to limit the scope of what the inventors regard as their invention. Nor is it intended to indicate that the following experiments are all or the only performed experiments. Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. Standard abbreviations can be used, for example, bp, base pair (s): kb, kilobase (s): pl, picoliter (s): s or sec, seconds (s) : Min, minutes (s): h or hr, hours (s): aa, amino acids (s): kb, kilobases (s): bp, base pairs (s): nt, nucleotides (s) Yes): i. m. Intramuscular (intramuscular): i. p. Intraperitoneal (intraperitoneal): s. c. , Subcutaneous (subcutaneous) and the like.

Example 1: Generation and Characterization of PD-L1 Mutants Materials and Methods Mutagenesis into PD-L1 Mouse full-length PD-L1 was cloned into the restriction enzyme sites of SacI and BamHI of the Clontech N1 mCherry vector. In order to improve localization and expression levels, the native leader peptide sequence was replaced by the EPO leader peptide sequence. Site-directed mutagenesis was performed using high fidelity KOD polymerase, 2 mM dNTPs, and 4 mM MgCl ₂ . The mutagenesis position was selected based on the crystal structure of the complex formed by human PD-L1 and PD-1 (PDB: 3BIK). Corresponding surface accessible positions in mouse PD-L1 were identified by taking a sequence alignment with human PD-L1 (36 positions in total). Mutagenesis was attempted such that each selected position was mutated to an Ala, Glu, or Arg residue. The overall success rate of mutagenesis was about 70%, and not all substitutions (A, E, and R) were obtained depending on the position. The expression of the sequence verified mutants was tested by transient gene transfer using 1 mL of HEK293 cell suspension. Thereafter, only mutants whose expression is equivalent to wild type PD-L1 and in which membrane localization occurs correctly are utilized in binding studies by microarray and FACS, resulting in 60 PD-L1 mutants for use in the assay. Got the final set including.

Microbead binding assay by FACS The PD-L1 mCherry mutant construct was transiently transfected into HEK293S cells and then loaded with protein A microbeads (Milltenyi). The protein A microbeads pre-saturated with a 4: 1 mixture of PD-1 Fc fusion and FITC-Fc protein were used. Based on a previous report by Genentech (16), the ratio of total Fc protein to beads was 5 ug per 10 uL of microbeads. FITC-Fc was used to give green fluorescence to normally non-fluorescent microbeads. For each titration experiment, 10 μg of fluorescein isothiocyanate (FITC) -Fc and 40 μg of either PD-1-Fc protein or B7-1-Fc protein, in a total volume of 5 mL of 1 × phosphate buffered saline ( PBS) was loaded onto 500 μL of protein A microbeads. The beads were incubated overnight (about 16 hours) at 4 ° C. The maximum storage period before using the loaded beads was 2 weeks. In the first experiment, the amount of loaded beads sufficient to saturate 150,000 cells transfected with wild-type PD-L1 (gene school transfer efficiency was consistently 60-70%) Was determined to be 75 μL. For the titration experiments, gene transfer of wild-type and mutated PD-L1 construct sets was performed in 24-well tissue culture plates containing 1 mL of HEK-293 cell suspension. Three days after gene transfer, the number of cells was counted, and the cells were diluted with 1 × PBS containing 2% BSA to obtain 1 × 10 ⁶ cells / mL. 150K cells (150 μL) were transferred to an Eppendorf tube and 75 μL of loaded microbeads were added along with an additional 1 × PBS (100 μL) containing 2% bovine serum albumin (BSA). Rotate the reaction and mix at 4 ° C for 1 hour, then add 4 ', 6-diamidino-2-phenylindole (DAPI) and analyze the sample immediately by flow cytometry on a BD Aria III cytometer did. Data analysis was performed by first gating live cells (DAPI negative) and then mCherry positive cells (PD-L1 expression). The proportion of FITC positive (microbead binding) mCherry positive cells was used as the “percentage binding”. For each experiment, the percent binding was normalized to wild type binding.

Purification of Recombinant Fc Fusion Protein To clone mPD-L1 Fc fusion protein, full-length wild-type or mutated PD-L1 ectodomain (residues F19-R237) was replaced with Fc with a his-tag at the C-terminus. Subcloned into LIC vector containing domain (mIgG2a-10 × His). These construct and isotope-only controls were transiently expressed in 1 L of HEK293 cell suspension. Four days after gene transfer, the medium was collected, pH was adjusted by adding 50 mM MES, and solubility was improved by adding 100 mM Arg-Cl (pH 7.6). Thereafter, the Fc fusion was purified with Ni ²⁺ -NTA resin (GE). This purification was performed by using a weight flow on a glass column (600 mL volume) packed with 10 mL total volume of resin after using a batch binding method. The Ni ²⁺ -nitroacetic acid (NTA) resin was added to 100 column volumes of wash buffer (50 mM MES (pH 6.5), 100 mM Arg-Cl, 5 mM imidazole, 150 mM NaCl, 10% Glycerol) and the bound protein was eluted with the same buffer containing 500 mM imidizole. The eluate from the nickel resin was concentrated and further purified by gel filtration on a S200 Sephadex column (GE) in 50 mM MES (pH 6.5), 100 mM Arg-Cl, 150 mM NaCl, 10% glycerol. Wild-type mPD-1 Fc (residues L25-Q167) and mB7-1 Fc (residues D37-K245) constructs were cloned into LIC vectors for lentiviral expression. This vector also contains the mIgG2a-10 × His tag. These constructs were transfected with the lentiviral packaging plasmid and the virus supernatant was collected 2 days later. Large scale transduction was initiated in 125 mL baffled flasks using 20 × 10 ⁶ cells and 5-10 mL of viral supernatant. On the third day from the transduction, the complete medium was changed, and on the fifth day, the culture was scaled up until the final volume reached 1.5 L. On day 12, the supernatant was collected for purification. PD-1 and B7-1 were obtained by refine | purifying the obtained lentiviral supernatant. This purification was performed as described for mPD-L1.

FACS titration assay A fluorescence activated cell sorting (FACS) titration assay was performed using PD-1 Fc fusion protein and B7-1 Fc fusion protein purified in-house as described above. HEK293 floating cells were transfected with wild-type or mutated PD-L1 constructs. Three days after gene transfer, the number of cells was counted, and the cells were diluted with 1 × PBS to give 1 × 10 ⁶ cells / mL. A premixed reaction containing a final concentration of 1 μM Fc fusion protein and 1.5 μM Alexa 488 goat anti-mouse secondary antibody was incubated on ice for 30 minutes. Thereafter, this premixed reaction solution was added to the wells of the 96-well plate while increasing the amount, and the volume was adjusted to 50 μL. Thereafter, 150 μL of diluted cells (total number of cells 150,000) was added to the wells. Binding was performed for 1 hour at 4 ° C., and the cells were washed 3 times with PBS by centrifugation and then analyzed by FACS. Live gated cells were partially gated with mCherry, and mCherry positive cells were partially gated with Alexa-488. Percentage binding indicates the percentage of mCherry cells that are positive for Alexa-488. Data are shown as the average of three independent experiments fitted against Y = B max * X / (EC ₅₀ + X), which is the equation for a single binding site.

T cell activation assay Spleens were collected from C57BL / 6 mice and CD4 ⁺ T cells were isolated using mouse anti-CD4 microbeads (Milltenyi). CD4 ⁺ T cells were collected in RPMI complete medium supplemented with 10% fetal bovine serum (FBS), penicillin / streptomycin antibiotics, 2 mM L-glutamine, and 0.1% BME. After counting cells and staining the cells with carboxyfluorescein N-succinimidyl ester (CFSE) (Invitrogen) using the manufacturer's protocol, the number of cells was counted again. On the same day, seed 75,000 cells per well in a 96-well TC plate and place in RPMI complete medium to leave it in an inactive state or with 33.3 nM (about 5 ug / mL) anti-CD3. 33.3 nM anti-CD3 in the presence of either activated or approximately 5-fold molar excess (174.3 nM) of control Fc protein, wild-type PD-L1-Fc protein, or mutant PD-L1 Fc protein Activated with. Four days after activation, proliferation was determined by FACS by performing a gate on non-activated T cells, thereby analyzing the dilution of CSFE. Data obtained from each experiment was normalized to a control Fc population and averaged a total of three independent experiments.

PD-1 / B7-1 competitive binding experiment The mB7-1 hIgG1Fc fusion construct was cloned. This construct used the same erythropoietin (EPO) leader, mB7-1 ectodomain boundary, and linker sequence as the original mIgG2a construct. This construct was expressed transiently in HEK293 cells and purified as described above for other Fc fusion proteins used. For competition experiments, transient gene transfer was performed on HEK293 floating cells using wild-type mPD-L1 mCherry. Three days after gene introduction, the number of gene-introduced cells was counted, and the cells were diluted to 1 × 10 ⁶ cells / mL. B7-1 hIgG1 fusion protein is added to 100,000 transgenic cells at a final dimer concentration of 5 nM, in the absence of PD-1 mIgG2a protein, or at a concentration of PD-1 mIgG2a protein It was present as an ascending series (dimer concentration 0.01-250 nM). As a parallel experiment, a titration of purified mIgG2a isotype control was performed at an equivalent molar concentration. Protein binding was performed on a 96-well plate shaker by shaking at 900 rpm and 22 ° C. for 1 hour. After binding, the plate was washed twice with 1 × PBS containing 0.2% BSA, anti-human (H + L) Alexa488-labeled secondary antibody (Invitrogen) was added at 0.01 μg / μL (1 μg total), Incubated for 30 minutes. The cells were then washed twice more with 1 × PBS containing 0.2% BSA. Samples were immediately analyzed by FACS, and data was gated on the percentage of cells that were positive for mCherry (FL4-PD-L1 expression) and also positive for Alexa488 (FL1-B7-1 binding). Competition data was normalized to 5 nM B7-1 binding in the absence of mPD-1 and plotted as a function of log [mPD-1]. The average data from 3 independent experiments was fitted using Y = minimum value + (maximum value−minimum value) / (1 + 10 ^{× -logEC50} ) where the competition site is one competition model equation.

Results Mechanistic analysis by microarray analysis In order to generate a selective PD-L1 reagent, the X-ray structure of the complex of PD-1 and PD-L1 was used as a framework, so The group was identified and, as a result, 36 solvent-exposed residues contained in the Ig variable domain of PD-L1 were identified (24). A series of characteristic physicochemical properties of the side chain were sampled by changing each residue to alanine, arginine, and glutamic acid. A cell microarray platform was first used to load a PD-1 or B7-1 Fc fusion protein against a series of wild-type and mutated PD-L1 constructs. According to these experiments, mutants affecting only PD-1 binding (D122A, Y123A, Y123R, K124A, K124D, R125A, R125D), mutants affecting only B7-1 binding (Y56A, Y56D, E72R, G119D, G120D). ), Or mutants (L53R, G119R, A121R) that affected both (Table 1 shown in FIG. 10). However, it has proved difficult to consistently quantify PD-1 / B7-1 binding using cell microarrays for the following reasons. (1) The affinity of B7-1 for PD-L1 is low, so the signal-to-noise ratio was reduced in these arrays compared to those loaded with PD-1, and (2) complete loss of binding. Although easy to identify, moderate reductions in binding often occurred in conjunction with the fact that they were more variable and (3) slides were independently printed, transduced, and processed The inherent variability of each slide adds to the variability of the signal-to-noise ratio, making direct comparisons more difficult.

Validation by FACS analysis To validate and more quantitatively evaluate the binding properties of PD-L1 variants, we performed a high-throughput fluorescence activated cell sorting (FACS) assay. In this assay, 96 samples can be examined about every 15 minutes. This FACs platform provides an improved dynamic range compared to cell microarrays. It is noteworthy that the display format of the search protein is modified. Although the bivalent Ig fusion used in the microarray platform is effective in identifying interactions with moderate affinity, weaker interactions may be missed. To aid in the detection of the wide range of apparent affinity predicted in the analysis of the library of PD-L1 variants, the valency increased by capture and presentation with magnetic microbeads was utilized (FIG. 7A). For example, in the search for a microarray presenting PD-L1, it was necessary to increase the concentration of B7-1 Fc compared to PD-1 Fc, which resulted in a stronger background signal. The dynamic range enhancement observed using the FACS microbead assay is due, at least in part, to the reduced background due to non-specific binding. There are two possible reasons for this: (1) No secondary antibody is used in the microbead assay, and (2) an increase in affinity means a decrease in the amount of protein that can be used for cell loading. Microbead assays have the added advantage of not requiring any washing steps, which minimizes the loss of bound sample and makes protein binding measurements in the assay much more direct. Furthermore, for some lower affinity interactions, such as the interaction between B7-1 and PD-L1, it is difficult to achieve saturation with soluble B7-1 Fc in the FACS assay. , B7-1 Fc-conjugated microbeads resulted in significant improvements.

  Briefly, 55 different mutant PD-L1-mCherry fusions that were surface displayed were individually and transiently transfected into the HEK293 cell line. The ability of these cells to bind to either wild-type PD-1 Ig fusion protein or FITC-loaded microbeads conjugated with wild-type B7-1 Ig fusion protein was explored by flow cytometry (FIG. 7B). Importantly, the transient protein expression levels for all of the mutants used in the analysis were similar to the wild type, so that these mutations caused an overall change in the structure or stability of PD-L1. There seems to be no letting it happen. In addition, examination of wild-type and mutant PD-L1 mutants with a fluorescence microscope showed that the C-terminal mCherry fusion protein was correctly localized to the membrane, indicating that the mutant protein was correctly folded and processed. Suggesting that it was inserted into the membrane. As a result of these tests, PD-L1 mutants specifically bound to PD-1 (D49R, V54D, V54R, Y56A, Y56D, Y56R, Q66D, E72R, G119D, G120D) or B7-1 were specifically bound. PD-L1 mutant (D122A, Y123R, Y123A, K124A, K124D, K124R, R125A, R125D), or PD-L1 mutant that did not bind to either PD-1 or B7-1 (L53D, L53R, I115D) , I116R, G119R, G120A, G120R, A121D, A121R, D122R). When the affected residues are mapped to the crystal structure of the complex of PD-1 and PD-L1, the PD-L1 surface responsible for PD-1 binding and B7-1 binding overlaps but is clearly different It has been shown. These results validated what was obtained by the first cellular microarray experiment, especially for mutants that were shown to significantly reduce but not eliminate binding to PD-1 or B7-1. More quantitative evaluations were obtained for 1 binding and B7-1 binding (Table 1 (FIG. 10), Table 2 (FIG. 11)). For example, in cell microarray experiments, the levels of PD-1 binding and B7-1 binding for V54D PD-L1 mutant and Q66D PD-L1 mutant were similar, but in the microbead FACS assay these same In two mutants, PD-1 binding was shown to be at the wild type level and B7-1 binding was significantly reduced.

  Sequence alignment analysis of PD-L1 and PD-L2 also provides clues to the relative importance of these residues for PD-1 binding and B7-1 binding. In general, residues specific for PD-1 binding are highly conserved in both PD-L1 and PD-L2, which predicts that both ligands bind to PD-1. The On the other hand, many of the residues identified as specifically binding to B7-1 are highly conserved only in PD-L1, but not in PD-L2, which indicates that PD-L2 is B7 It also makes sense to not bind to -1. This supports data highlighting the importance of V54 and Y56 for B7-1 binding.

Biological activity of PD-L1 variants in T cell proliferation assays High-throughput transient gene transfer of HEK293 cells in 24-well suspension tissue culture plates produces recombinant secreted Fc fusion protein in an amount commensurate with screening. Optimized to produce. Using this method, Fc fusion proteins were purified on a subset of PD-L1 variants with altered binding properties. After nickel purification of the PD-L1 protein on a small scale, analytical gel filtration showed that the behavior of the selected mutant was similar to the wild type protein. Prior to testing activity in the T cell proliferation test, the quality of each mutant protein was assessed by FACS analysis for binding to HEK cells expressing surface immobilized PD-1 or B7-1 (GFP fusion), As a result, it was confirmed that the behavior of the soluble reagent was as expected (that is, PD-L1_Y56A_Fc binds to cells expressing PD-1 but not to cells expressing B7-1). .

In vitro T cell activation assays were used to characterize the biological activity of functionally probed PD-L1 variants. This is a widely used assay, in which a plate-bound anti-CD3 antibody is used to stimulate T cell activity via the T cell receptor. Anti-CD3 was simultaneously immobilized on plates in the presence of either IgG control, wild type PD-L1, or PD-L1 mutant, and activation of primary CD4 ⁺ mouse T cells labeled with CSFE was measured. In a situation where CD4 ⁺ T cells are activated in an anti-CD3-mediated manner, wild-type PD-L1 suppressed activation compared to the isotype control (FIG. 8D), and PD- decreased in the level of PD-1 binding. The L1 mutant was shown to have a markedly reduced ability to suppress T cell activation. In contrast, PD-L1 mutants with reduced B7-1 activity elicited effects similar to wild-type PD-L1. These data indicate that under the in vitro experimental system used, PD-L1-induced suppression of CD4 ⁺ T cell activation occurs primarily through the interaction of PD-L1 with PD-1. Suggests. These data indicate that certain organisms that can help define the distinct contributions of PD-L1 and PD-1 interactions and PD-L1 and B7-1 interactions to mammalian immunity. This shows the feasibility of generating mutants having biological activity.

Competition of PD-1 with B7-1 for binding to PD-L1 According to the mutagenesis data, the binding surface of PD-L1 binding to PD-1 and B7-1 is overlapping, but it is clearly different This indicates that PD-1 and B7-1 will compete for binding to PD-L1. This hypothesis was tested using a cell-based FACS competition assay utilizing the B7-1 and PD-1 Fc fusion proteins we purified using two different Fc fusion isotypes. Using these reagents, titration was performed in a series of increasing concentrations of PD-1 (mIgG2a), and at the same time, disappearance of B7-1 (hIgG1) bound to HEK cells expressing wild-type PD-L1 was determined as anti-human. It was selectively monitored using Alexa 488 secondary antibody (FIG. 9A). The results show that PD-1 binding effectively replaces B7-1 from cells expressing PD-L1, which is based on the finding based on PD-L1 mutagenesis that the binding sites overlap. Is to support.

7A-7C: Screening of PD-L1 mutants using high-throughput microbead binding assay by FACS A) Schematic representation of FACS microbead binding assay. B) A representative control microbead experiment is shown. Microbeads conjugated with control Fc, PD-1 Fc fusion protein, or B7-1 Fc fusion protein were loaded on cells expressing either mCherry alone (-control) or PD-L1 mCherry. The FACS data was obtained by gating all living cells, and PD-1-coated microbeads and B7-1 were coated on cells expressing wild-type PD-L1. Shows that both microbeads bind (top right quadrant). C) Microbead binding data by FACS for a panel of 54 PD-L1 mutants. Data show the fraction of mCherry positive cells (PD-L1 expression) bound to microbeads coated with either PD-1 (blue) or B7-1 (red), binding to wild type It has been standardized. PD-1 binding and B7-1 binding were performed in parallel in triplicate experiments, error bars indicate standard deviation.

8A-8D: Characterization of PD-L1 variants with altered binding to PD-1 or B7-1 A) Crystal structure of the complex of PD-1 and PD-L1 (PDB: 3SBW), PD -Only the IgC and IgV domains of L1 are shown. The IgV domain has been expanded, and residues whose binding has changed when mutated are labeled and colored according to the following. Green = PD-1 binding is affected, red = B7-1 binding is affected, gray = both PD-1 and B7-1 binding are affected. B) Data obtained from FACS titration experiments in which cells expressing either wild type PD-L1 or mutants were titrated with increasing concentrations of recombinant PD-1 Fc fusion protein or B7-1 Fc fusion protein. Show. Binding was detected using an anti-mouse Alexa 488 secondary antibody. Data points represent the average of three independent experiments and error bars represent standard deviation. The curve shows a fit of the data to the binding model with a single binding site. C) shows a table of EC ₅₀ values and B max values obtained from FACS titration experiments in B. An asterisk indicates that the titration is very weak (baseline) and the data could not be fit. D) Data are CD4 isolated from C57BL / 6 mice and labeled with CSFE and activated after 4 days stimulation with anti-CD3 in the presence of isotype control, wild type, or mutated PD-L1 Fc fusion protein. ⁺ T cell fractions are shown. Activation is normalized to the isotype control and represents three independent experiments.

9A-9B: Competition of PD-1 with B7-1 for binding to PD-L1 A) Schematic representation of the competition assay. Briefly, HEK293 cells transiently transfected with PD-L1 mCherry were incubated with mB7-1 hIgG1 protein in the absence or presence of increasing concentrations of mPD-1 mIgG2a. Subsequently, the amount of mB7-1 hIgG1 bound to the cells was monitored by FACS analysis using anti-human Alexa488 antibody. B) Shows a heat map showing results from one representative experiment. In the presence of control mIgG2a, no loss of binding of mB7-1 hIgG1 was observed. The graph shows the mean and standard deviation for data obtained from three independent experiments. This data was fitted using a model equation with a single competitor site in the Prism software and the EC ₅₀ was calculated as 8.3 ± 1.5 nM.

Example 2: In vivo activity of PD-L1 / synTac In the NY8.3 TCR transgenic NOD model of type 1 diabetes (NOD 8.3), pancreatic beta cells are associated with the MHC class I allele H-2K ^d Invasive T cell autoimmunity occurs that targets the antigen Igrp 206-214. NOD8.3 mice have a high frequency of circulating transgenic (Igrp-specific) T cells. To determine the effect on the frequency of pathogenic transgenic T cells in the spleen, the NOD8.3 mouse PD-L1 / synTac having Igrp _206-214 / H-2K ^d and PD-L1 (G119R variant) Administered. As shown in FIG. 12, PD-L1 (G119R) / synTac causes a dose-dependent depletion of T cells specific for Igrp _206-214 / H-2K ^d , but on non-specific T cells. Does not have this effect.

  Although the invention has been described with reference to specific embodiments thereof, those skilled in the art will recognize that various changes can be made and replaced with equivalents without departing from the true spirit and scope of the invention. I want to be. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, method, or method step (s) to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

This application claims the benefit of US Provisional Patent Application No. 62 / 338,128, filed May 18, 2016, which is hereby incorporated by reference in its entirety.

Claims (87)

A mutant PD- comprising an amino acid sequence having at least 85% amino acid sequence identity to the PD-L1 amino acid sequence shown in FIG. 2A or FIG. 2B or the PD-L1 amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 An L1 immunoregulatory polypeptide comprising:
The mutant PD-L1 immunomodulatory polypeptide has one or more amino acid substitutions compared to the PD-L1 amino acid sequence shown in FIG. 2A or the PD-L1 amino acid sequence shown in SEQ ID NO: 1.
a) Binding affinity of the PD-L1 amino acid sequence shown in FIG. 2A or FIG. 2B to the PD1 polypeptide having the amino acid sequence shown in FIG. 3A or FIG. 3B, or PD- FIG. 3 shows a reduced binding affinity for the PD1 polypeptide compared to the binding affinity of the L1 amino acid sequence, and / or b) for a B7-1 polypeptide having the amino acid sequence shown in FIG. 3C or FIG. 3D. Binding to the B7-1 polypeptide as compared to the binding affinity of the PD-L1 amino acid sequence shown in 2A or 2B or the binding affinity of the PD-L1 amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. Mutant PD-L1 immunoregulatory polypeptide that exhibits reduced affinity.   The polypeptide comprises a substitution of amino acid D26, amino acid T37, amino acid V54, amino acid Q66, or amino acid E72 based on the amino acid numbering shown in FIG. 2. The variant immune modulatory polypeptide of claim 1, comprising a substitution of amino acid D26, amino acid T37, amino acid I54, amino acid Q66, or amino acid E72 based on numbering.   The mutant immunoregulatory polypeptide has a binding affinity for the PD1 polypeptide as shown in FIG. 2A or 2B or a PD-L1 polypeptide comprising the PD-L1 amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. 3. The variant immunoregulatory polypeptide according to claim 1 or claim 2, wherein the variant immunomodulating polypeptide is less than 10% to less than 75% of the binding affinity.   The mutant immunoregulatory polypeptide has a PD-L1 polypeptide whose binding affinity to the B7-1 polypeptide comprises the PD-L1 amino acid sequence shown in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2. The mutant immunoregulatory polypeptide according to any one of claims 1 to 3, which has a binding affinity of less than 10% to less than 75%.   The mutant immunoregulatory polypeptide has a binding affinity for the PD1 polypeptide as shown in FIG. 2A or 2B or a PD-L1 polypeptide comprising the PD-L1 amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. 5. The variant immunomodulatory polypeptide of any one of claims 1, 2, or 4, wherein the variant has a binding affinity of 75% to greater than 95%.   The mutant immunoregulatory polypeptide has a PD-L1 polypeptide whose binding affinity to the B7-1 polypeptide comprises the PD-L1 amino acid sequence shown in FIG. 2A or FIG. 2B or SEQ ID NO: 1 or SEQ ID NO: 2. The variant immunomodulatory polypeptide according to any one of claims 1 to 3 or claim 5, which has a binding affinity of 75% to more than 95%. a)
i) an epitope,
ii) a first polypeptide comprising a first major histocompatibility complex (MHC) polypeptide in N-terminal to C-terminal order;
b)
i) a second MHC polypeptide, and ii) an optional immunoglobulin (Ig) Fc polypeptide or a second polypeptide comprising a non-Ig backbone in N-terminal to C-terminal order;
A multimeric polypeptide comprising:
The multimeric polypeptide comprises one or more immunoregulatory domains, wherein at least one of the one or more immunoregulatory domains is
A) the C-terminus of the first polypeptide,
B) the N-terminus of the second polypeptide,
C) located at the C-terminus of the second polypeptide, or D) at the C-terminus of the first polypeptide and at the N-terminus of the second polypeptide,
The immunoregulatory domain is an amino acid having at least 85% amino acid sequence identity to the PD-L1 amino acid sequence shown in FIG. 2A or FIG. 2B or the PD-L1 amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. Contains an array,
The mutant PD-L1 immunoregulatory polypeptide has one or more amino acid substitutions compared to the PD-L1 amino acid sequence shown in FIG. 2A or the PD-L1 amino acid sequence shown in SEQ ID NO: 1.
The mutant PD-L1 immunoregulatory polypeptide is:
a) Binding affinity of the PD-L1 amino acid sequence shown in FIG. 2A or FIG. 2B to the PD1 polypeptide having the amino acid sequence shown in FIG. 3A or FIG. 3B, or PD- shown in SEQ ID NO: 1 or SEQ ID NO: 2. FIG. 3 shows a reduced binding affinity for the PD1 polypeptide compared to the binding affinity of the L1 amino acid sequence, and / or b) for a B7-1 polypeptide having the amino acid sequence shown in FIG. 3C or FIG. 3D. Binding to the B7-1 polypeptide as compared to the binding affinity of the PD-L1 amino acid sequence shown in 2A or 2B or the binding affinity of the PD-L1 amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. A multimeric polypeptide that exhibits reduced affinity.   The multimeric polypeptide comprises a control multimeric polypeptide comprising an immunoregulatory domain comprising the PD-L1 amino acid sequence shown in FIG. 2A or FIG. 2B relative to a PD1 polypeptide having the amino acid sequence shown in FIG. Binding affinity or binding affinity for said PD1 polypeptide as compared to the binding affinity of a control multimeric polypeptide comprising an immunomodulatory domain comprising the PD-L1 amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. The multimeric polypeptide according to claim 7, which exhibits a decrease. The multimeric polypeptide is
a)
i) the epitope,
ii) said first MHC polypeptide, and iii) a first polypeptide comprising said immunoregulatory domain in N-terminal to C-terminal order;
b)
i) the second MHC polypeptide, and ii) the second polypeptide comprising the IgFc polypeptide in N-terminal to C-terminal order;
The multimeric polypeptide of claim 7, comprising: The multimeric polypeptide is
a)
a first polypeptide comprising i) said epitope, and ii) said first MHC polypeptide in order from N-terminus to C-terminus;
b)
i) the immunoregulatory domain,
iii) the second MHC polypeptide, and ii) the IgFc polypeptide,
A second polypeptide comprising, in order from the N-terminus to the C-terminus;
The multimeric polypeptide of claim 7, comprising: The multimeric polypeptide is
a)
a first polypeptide comprising i) said epitope, and ii) said first MHC polypeptide in order from N-terminus to C-terminus;
b)
i) said second MHC polypeptide,
ii) said IgFc polypeptide, and iii) a second polypeptide comprising said immunoregulatory domain in N-terminal to C-terminal order;
The multimeric polypeptide of claim 7, comprising: The multimeric polypeptide is
a)
a first polypeptide comprising i) said epitope, and ii) said first MHC polypeptide in order from N-terminus to C-terminus;
b)
i) the second MHC polypeptide, and ii) the second polypeptide comprising the immunoregulatory domain in the order N-terminal to C-terminal;
The multimeric polypeptide of claim 7, comprising: The multimeric polypeptide is
a)
a first polypeptide comprising i) said epitope, and ii) said first MHC polypeptide in order from N-terminus to C-terminus;
b)
a second polypeptide comprising: i) said immunoregulatory domain; and ii) said second MHC polypeptide in order from N-terminus to C-terminus;
The multimeric polypeptide of claim 7, comprising: The multimeric polypeptide is
a)
i) the epitope,
ii) said first MHC polypeptide, and iii) a first polypeptide comprising said immunoregulatory domain in N-terminal to C-terminal order;
b)
i) a second polypeptide comprising the second MHC polypeptide in N-terminal to C-terminal order;
The multimeric polypeptide of claim 7, comprising:   8. The multimeric polypeptide of claim 7, wherein the non-Ig backbone is an XTEN polypeptide, transferrin polypeptide, elastin-like polypeptide, silk-like polypeptide, or silk-elastin-like polypeptide.   16. The first MHC polypeptide according to any one of claims 7 to 15, wherein the first MHC polypeptide is a β2-microglobulin polypeptide and the second MHC polypeptide is an MHC class I heavy chain polypeptide. Multimeric polypeptide.   17. The multimeric polypeptide of claim 16, wherein the β2-microglobulin polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to one of the amino acid sequences shown in FIG.   17. The multimeric polypeptide of claim 16, wherein the MHC class I heavy chain polypeptide is an HLA-A, HLA-B, or HLA-C heavy chain.   19. The multimer of claim 18, wherein the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence shown in one of FIGS. 5A-5C. Polypeptide.   17. The first MHC polypeptide is an MHC class II alpha chain polypeptide, and the second MHC polypeptide is an MHC class II beta chain polypeptide. The multimeric polypeptide.   21. The multimeric polypeptide according to any one of claims 7 to 20, wherein the epitope is a T cell epitope.   A Fc polypeptide, wherein the IgFc polypeptide is an IgGl Fc polypeptide, IgG2 Fc polypeptide, IgG3 Fc polypeptide, IgG4 Fc polypeptide, IgAFc polypeptide, or IgMFc polypeptide. The multimeric polypeptide according to any one of the above.   24. The multimeric polypeptide of claim 21, wherein the IgFc polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence shown in FIGS.   23. The multimeric polypeptide according to any one of claims 7 to 22, wherein the first polypeptide and the second polypeptide are bound by a non-covalent bond.   The multimeric polypeptide according to any one of claims 7 to 22, wherein the first polypeptide and the second polypeptide are linked by a covalent bond.   The multimeric polypeptide according to claim 24, wherein the covalent bond is via a disulfide bond.   The first MHC polypeptide, or the linker between the epitope and the first MHC polypeptide, provides an amino acid substitution to provide a first Cys residue, and the second MHC polypeptide comprises: 26. The macromolecule of claim 25, comprising an amino acid substitution to provide a second Cys residue, wherein the disulfide bond is formed between the first Cys residue and the second Cys residue. Body polypeptide.   15. A multimeric polypeptide according to any one of claims 7 to 14, comprising a first linker inserted between the epitope and the first MHC polypeptide.   Said mutant PD-L1 immunomodulating polypeptide is selected from D26, T37, I54, Y56, Q66, E72, I115, G119, or G120 based on the amino acid numbering of the PD-L1 amino acid sequence shown in FIG. 2B 15. A multimeric polypeptide according to any one of claims 7 to 14, comprising an amino acid substitution.   29. A multimeric polypeptide according to any one of claims 7 to 28, comprising two or more mutated PD-L1 immunomodulatory polypeptides.   30. The multimeric polypeptide of claim 29, wherein the two or more immunomodulatory polypeptides are present in tandem.   The multimeric polypeptide according to any one of claims 7 to 28, wherein the immunomodulating polypeptide is the immunomodulating polypeptide according to any one of claims 1 to 6.   The multimeric polypeptide comprises a third polypeptide, wherein the third polypeptide is at least 90% amino acids relative to the immunomodulatory polypeptide of the first polypeptide or the second polypeptide. 32. A multimeric polypeptide according to any one of claims 29 to 31 comprising an immunomodulatory polypeptide comprising an amino acid sequence having sequence identity.   34. The multimeric polypeptide of claim 32, wherein the third polypeptide is covalently linked to the first polypeptide. The second polypeptide is
i) said second MHC polypeptide,
34. The multimeric polypeptide of any one of claims 7-13 and 15-33, comprising ii) said IgFc polypeptide, and iii) an affinity tag in order from N-terminus to C-terminus. A nucleic acid comprising a nucleotide sequence encoding a recombinant polypeptide comprising:
i) The recombinant polypeptide is
a) an epitope,
b) a first major histocompatibility complex (MHC) polypeptide,
c) an immunoregulatory polypeptide which is a mutant immunoregulatory polypeptide according to any one of claims 1-6;
d) a linker or ribosome skipping signal cleavable by proteolysis,
e) a second MHC polypeptide, and f) an immunoglobulin (Ig) Fc polypeptide in N-terminal to C-terminal order, or ii) the recombinant polypeptide
a) an epitope,
b) a first MHC polypeptide,
c) a linker or ribosome skipping signal cleavable by proteolysis,
d) an immunoregulatory polypeptide which is a mutant immunoregulatory polypeptide according to any one of claims 1-6;
e) a nucleic acid comprising a second MHC polypeptide, and f) an IgFc polypeptide in N-terminal to C-terminal order.   36. The nucleic acid of claim 35, wherein the first MHC polypeptide is a β2-microglobulin polypeptide and the second MHC polypeptide is an MHC class I heavy chain polypeptide.   37. The nucleic acid of claim 36, wherein the β2-microglobulin polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to one of the amino acid sequences shown in FIG.   37. The nucleic acid of claim 36, wherein the MHC class I heavy chain polypeptide is an HLA-A, HLA-B, or HLA-C heavy chain.   39. The nucleic acid of claim 38, wherein the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence shown in any one of Figures 5A-5C.   36. The nucleic acid of claim 35, wherein the first MHC polypeptide is an MHC class II alpha chain polypeptide and the second MHC polypeptide is an MHC class II beta chain polypeptide.   36. The nucleic acid of claim 35, wherein the epitope is a T cell epitope.   36. The nucleic acid of claim 35, wherein the IgFc polypeptide is an IgGl Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgAFc polypeptide, or an IgMFc polypeptide.   43. The nucleic acid of claim 42, wherein the IgFc polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence shown in FIGS.   The mutant PD-L1 polypeptide comprises a substitution of amino acid D26, amino acid T37, amino acid V54, amino acid Q66, or amino acid E72 based on the amino acid numbering shown in FIG. 2A, or the polypeptide is shown in FIG. 2B. 36. The nucleic acid of claim 35, comprising a substitution of amino acid D26, amino acid T37, amino acid I54, amino acid Q66, or amino acid E72 based on the indicated amino acid numbering.   36. The nucleic acid of claim 35, wherein the multimeric polypeptide comprises two or more mutant PD-L1 immunomodulatory polypeptides. The proteolytically cleavable linker or ribosome skipping signal is
a) LEVLFQGP (SEQ ID NO: 34),
b) ENLYTQS (SEQ ID NO: 35),
c) Furin cleavage site,
d) LVPR (SEQ ID NO: 37),
e) GSGATNFSLLKQAGDVEENGPP (SEQ ID NO: 38),
f) GSGEGRGSLLTCGDVEENGPP (SEQ ID NO: 39),
g) GSGQCTNYALLKLAGDVESPNPGP (SEQ ID NO: 40), and h) GSGVKQTLNFDLLKLAGDVESPNPGP (SEQ ID NO: 41)
36. The nucleic acid of claim 35, comprising an amino acid sequence selected from. The recombinant polypeptide is
a) a first leader peptide,
b) the epitope,
c) said first MHC polypeptide,
d) said immunomodulatory polypeptide,
e) a linker or ribosome skipping signal cleavable by said proteolysis,
f) a second leader peptide,
36. The nucleic acid of claim 35, comprising g) said second MHC polypeptide, and h) said immunoglobulin (Ig) Fc polypeptide in N-terminal to C-terminal order.   48. The nucleic acid of claim 47, wherein the first leader peptide and the second leader peptide are β2-M leader peptides.   36. The nucleic acid of claim 35, wherein the nucleotide sequence is operably linked to a transcriptional control element.   50. The nucleic acid of claim 49, wherein the transcriptional control element is a promoter that functions in eukaryotic cells.   The first MHC polypeptide, or the linker between the epitope and the first MHC polypeptide, provides an amino acid substitution to provide a first Cys residue, and the second MHC polypeptide comprises: An amino acid substitution is included to provide a second Cys residue, wherein the first Cys residue and the second Cys residue are between the first MHC polypeptide and the second MHC polypeptide. 36. The nucleic acid of claim 35, wherein said nucleic acid produces a disulfide bond.   A recombinant expression vector comprising the nucleic acid according to any one of claims 35 to 51.   53. The recombinant expression vector of claim 52, wherein the vector is a viral vector or a non-viral vector.   53. A host cell genetically modified with the recombinant expression vector of claim 52.   55. The host cell of claim 54, wherein the host cell is in vitro.   55. The host cell of claim 54, wherein the host cell is genetically modified such that the cell does not produce endogenous MHCβ2-microglobulin polypeptide.   55. The host cell of claim 54, wherein the host cell is a T lymphocyte. a)
i) an epitope,
a first polypeptide comprising ii) a first MHC polypeptide, and iii) an immunoregulatory domain which is a mutant immunoregulatory polypeptide according to any one of claims 1 to 6 in the order of N-terminal to C-terminal A first nucleic acid comprising a nucleotide sequence encoding
b)
a second nucleic acid comprising a nucleotide sequence encoding a second polypeptide comprising i) a second MHC polypeptide, and ii) an IgFc polypeptide in N-terminal to C-terminal order;
A composition comprising: a)
a first nucleic acid comprising a nucleotide sequence encoding i) an epitope, and ii) a first polypeptide comprising a first MHC polypeptide in N-terminal to C-terminal order;
b)
i) an immunoregulatory domain that is a mutant immunoregulatory polypeptide according to any one of claims 1 to 6; ii) a second MHC polypeptide; and iii) an IgFc polypeptide in the order N-terminal to C-terminal A second nucleic acid comprising a nucleotide sequence encoding a second polypeptide;
A composition comprising:   60. The composition of claim 58 or claim 59, wherein the first nucleic acid and / or the second nucleic acid is present in a recombinant expression vector.   61. A host cell genetically modified with the composition of any one of claims 58-60. A method for producing a multimeric polypeptide according to any one of claims 7 to 34, comprising:
a) in vitro culture of the host cell in a medium under conditions in which the host cell of any one of claims 54-57 and 61 synthesizes the multimeric polypeptide;
b) isolation of the multimeric polypeptide from the host cell and / or the medium;
Including a method.   The second polypeptide comprises an affinity tag, and the isolation comprises contacting the multimeric polypeptide produced by the cell with a binding partner for the affinity tag, wherein the binding partner is 64. The method of claim 62, wherein the multimeric polypeptide is immobilized by immobilization.   64. The method of claim 62, comprising elution of the immobilized multimeric polypeptide. A method for selectively modulating the activity of epitope-specific T cells, comprising:
Comprising contacting the epitope-specific T cell with the multimeric polypeptide of any one of claims 7 to 34,
Wherein said contacting selectively modulates the activity of said epitope-specific T cells.   66. The method of claim 65, wherein the immunomodulating polypeptide is a suppressor polypeptide and the multimeric polypeptide suppresses the epitope-specific T cells.   66. The method of claim 65, wherein the contacting is in vitro.   66. The method of claim 65, wherein the contacting is in vivo.   66. The method of claim 65, wherein the contact is ex vivo. A method of selectively modulating the activity of epitope-specific T cells in an individual comprising:
35. A method comprising administering to said individual an effective amount of a multimeric polypeptide according to any one of claims 7 to 34 effective for selective modulation of the activity of epitope-specific T cells in said individual.   72. The method of claim 70, wherein the immunomodulatory polypeptide is a suppressor polypeptide and the multimeric polypeptide suppresses the activity of the epitope-specific T cells.   The method according to claim 71, wherein the epitope is a self-epitope, and the administration selectively suppresses the activity of T cells specific to the self-epitope.   The method according to claim 71, wherein the epitope is an epitope present in an allograft, and the administration selectively suppresses the activity of T cells specific for the allograft. A method of treating an autoimmune disorder in an individual comprising:
35. Administration of the multimeric polypeptide of any one of claims 7-34 in an effective amount to the individual in an effective amount effective for selective suppression of T cell activity specific for the self-epitope in the individual. The method whereby the autoimmune disorder is treated.   75. The method of claim 74, wherein the autoimmune disease is multiple sclerosis, systemic lupus erythematosus, or autoimmune arthritis. A method for suppressing allograft rejection in an individual comprising:
35. An effective amount of a multimeric polypeptide according to any one of claims 7 to 34 effective for selective suppression of T cell activity specific for an epitope present in an allograft present in said individual. A method comprising administration to said individual, whereby allograft rejection is suppressed.   77. The method of claim 76, wherein the allograft is a kidney, lung, skin, liver, bone, cartilage, or heart.   78. The method according to any one of claims 70 to 77, wherein the administration is subcutaneous administration.   78. The method of any one of claims 70 to 77, wherein the administration is intravenous administration.   78. A method according to any one of claims 70 to 77, wherein the administration is intramuscular.   78. The method of any one of claims 70 to 77, wherein the administration is systemic administration.   78. The method of any one of claims 70 to 77, wherein the administration is at a location remote from the treatment site.   78. The method of any one of claims 70 to 77, wherein the administration is topical administration.   78. The method of any one of claims 70 to 77, wherein the administration is at or near a treatment site. A composition comprising: a) a multimeric polypeptide according to any one of claims 7 to 34; and b) a pharmaceutically acceptable excipient. A composition comprising: a) a nucleic acid according to any one of claims 35 to 51, or a recombinant expression vector according to claim 52 or 53, and b) a pharmaceutically acceptable excipient. .